Water-soluble polymeric dyes having pendant chromophores

ABSTRACT

Water soluble light harvesting multichromophores having pendant chromophore groups are provided. The light harvesting multichromophore has a polymeric backbone including non-conjugated repeat units and a plurality of pendant donor chromophore groups linked to a non-conjugated repeat unit of the polymeric backbone. A pendant chromophore group can be a BODIPY group substituted with one or more water soluble groups. Polymeric tandem dyes based on the subject multichromophores are provided that further include an acceptor fluorophore linked to a non-conjugated repeat unit of the polymeric backbone and configured in energy-receiving proximity to a pendant donor chromophore group. Also provided are labelled specific binding members that include the subject polymeric tandem dyes. Methods of evaluating a sample for a target analyte and methods of labelling a target molecule in which the subject polymeric tandem dyes find use are provided. Systems and kits for practicing the subject methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

Pursuant to 35 U.S.C. § 119(e), this is a continuation application ofU.S. patent application Ser. No. 16/368,513, filed Mar. 28, 2019, whichclaims priority to U.S. Provisional Patent Application Ser. No.62/650,935, filed Mar. 30, 2018, and U.S. Provisional Patent ApplicationSer. No. 62/715,722, filed Aug. 7, 2018; the disclosures of whichapplications are incorporated herein by reference.

INTRODUCTION

Fluorescent dyes are compounds which, when irradiated with light of awavelength which they absorb, emit light of a (usually) differentwavelength. Fluorescent dyes find use in a variety of applications inbiochemistry, biology and medicine, e.g. in diagnostic kits, inmicroscopy or in drug screening. Fluorescent dyes are characterized by anumber of parameters allowing a user to select a suitable dye dependingon the desired purpose.

Parameters of interest include the excitation wavelength maximum, theemission wavelength maximum, the Stokes shift, the extinctioncoefficient, the fluorescence quantum yield and the fluorescencelifetime. Dyes may be selected according to the application of interestin order to, e.g., allow penetration of exciting radiation intobiological samples, to minimize background fluorescence and/or toachieve a high signal-to-noise ratio.

Molecular recognition involves the specific binding of two molecules.Molecules which have binding specificity for a target biomolecule finduse in a variety of research and diagnostic applications, such as thelabelling and separation of analytes, flow cytometry, in situhybridization, enzyme-linked immunosorbent assays (ELISAs), western blotanalysis, magnetic cell separations and chromatography. Targetbiomolecules may be detected by labelling with a fluorescent dye.

SUMMARY

Water soluble light harvesting multichromophores having a plurality ofpendant chromophore groups are provided. The light harvestingmultichromophore has a polymeric backbone including non-conjugatedrepeat units and a plurality of pendant donor chromophore groups eachindependently linked to a non-conjugated repeat unit of the polymericbackbone. A pendant chromophore group can be a BODIPY group substitutedwith one or more water soluble groups. Polymeric tandem dyes based onthe subject multichromophores are also provided that further include anacceptor fluorophore linked to a non-conjugated repeat unit of thepolymeric backbone and configured in energy-receiving proximity to atleast one pendant donor chromophore group of the light harvestingmultichromophore. Also provided are labelled specific binding membersthat include the subject polymeric tandem dyes. Methods of evaluating asample for the presence of a target analyte and methods of labelling atarget molecule in which the subject polymeric tandem dyes find use arealso provided. Systems and kits for practicing the subject methods arealso provided.

BRIEF DESCRIPTION OF THE FIGURES

It is understood that the drawings, described below, are forillustration purposes only. The drawings are not intended to limit thescope of the present teachings in any way.

FIG. 1 depicts the general structure of an exemplary polymeric tandemdye where the “Dye” is an acceptor fluorophore.

FIG. 2 shows a normalized absorption spectrum for an exemplarymultichromophore and emission spectra for a series of polymeric tandemdyes including the multichromophore.

FIG. 3 shows emission spectra for an exemplary multichromophore and aseries of polymeric tandem dyes including the multichromophore, all at0.04 OD absorption.

FIG. 4 depicts the structure of an exemplary polymeric tandem dye havinga peptidic backbone. D is a BODIPY donor pendant group, A is an acceptordye and biolinker is a linker including a chemoselective functionalgroup for attachment of the tandem dye to a biomolecule.

FIG. 5A shows absorption and emission spectra for the BODIPY donorpendant group that was used to prepare the multichromophore of FIG. 4.FIG. 5B shows absorption and emission spectra for the acceptor dye usedto prepare the multichromophore of FIG. 4. FIG. 5C shows absorption andemission spectra for the exemplary polymeric tandem dye of FIG. 4.

FIG. 6A illustrates homo-energy transfer between pendant donorchromophores which leads preferentially to continued reversible energytransfer amongst equal chromophores rather than emission from a singlechromophore. This process can result in self-quenching and quantumyields that are significantly lower than those observed for a singleisolated chromophore. FIG. 6B illustrates hetero-energy transfer whichleads to primarily one way energy transfer between differentchromophores. Energy transfer to the secondary chromophore leadspreferentially to emission, limited by the quantum yield of the acceptorand a single donor chromophore.

FIG. 7A-7B illustrate synthetic schemes for the preparation of exemplarymultichromophores having a linked BODIPY dye (“Dye”) (FIG. 7A) andpolymeric tandem dye (FIG. 7B) using click polymerization methods. “Dye”and “Donor” can refer to a donor dye such as a BODIPY and “Acceptor” isan acceptor fluorophore.

FIG. 8 show the absorption and emission spectra of an exemplarymultichromophore of FIG. 7A that includes linked pendant BODIPY dyes.

FIG. 9A-9B show the absorption (FIG. 9A) and emission spectra (FIG. 9B)of a series of exemplary polymeric tandem dyes including linked pendantBODIPY donor dyes and a variety of acceptor dyes having differentemission maximum wavelengths.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Still, certain terms aredefined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a dye” refersto one or more dyes, i.e., a single dye and multiple dyes. It is furthernoted that the claims can be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

As used herein, the terms “chemoselective functional group” and“chemoselective tag” are used interchangeably and refer to a functionalgroup that can selectively react with another compatible functionalgroup to form a covalent bond, in some cases, after optional activationof one of the functional groups. Chemoselective functional groups ofinterest include, but are not limited to, thiols and maleimide oriodoacetamide, amines and carboxylic acids or active esters thereof, aswell as groups that can react with one another via Click chemistry,e.g., azide and alkyne groups (e.g., cyclooctyne groups), tetrazine,transcyclooctene, dienes and dienophiles, and azide, sulfur(VI) fluorideexchange chemistry (SuFEX), sulfonyl fluoride, as well as hydroxyl,hydrazido, hydrazino, aldehyde, ketone, azido, alkyne, phosphine,epoxide, and the like.

As used herein, the term “sample” relates to a material or mixture ofmaterials, in some cases in liquid form, containing one or more analytesof interest. In some embodiments, the term as used in its broadestsense, refers to any plant, animal or bacterial material containingcells or producing cellular metabolites, such as, for example, tissue orfluid isolated from an individual (including without limitation plasma,serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) orfrom in vitro cell culture constituents, as well as samples from theenvironment. The term “sample” may also refer to a “biological sample”.As used herein, the term “a biological sample” refers to a wholeorganism or a subset of its tissues, cells or component parts (e.g. bodyfluids, including, but not limited to, blood, mucus, lymphatic fluid,synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amnioticcord blood, urine, vaginal fluid and semen). A “biological sample” canalso refer to a homogenate, lysate or extract prepared from a wholeorganism or a subset of its tissues, cells or component parts, or afraction or portion thereof, including but not limited to, plasma,serum, spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors and organs. In certain embodiments, the sample hasbeen removed from an animal or plant. Biological samples may includecells. The term “cells” is used in its conventional sense to refer tothe basic structural unit of living organisms, both eukaryotic andprokaryotic, having at least a nucleus and a cell membrane. In certainembodiments, cells include prokaryotic cells, such as from bacteria. Inother embodiments, cells include eukaryotic cells, such as cellsobtained from biological samples from animals, plants or fungi.

The terms “support bound” and “linked to a support” are usedinterchangeably and refer to a moiety (e.g., a specific binding member)that is linked covalently or non-covalently to a support of interest.Covalent linking may involve the chemical reaction of two compatiblefunctional groups (e.g., two chemoselective functional groups, anelectrophile and a nucleophile, etc.) to form a covalent bond betweenthe two moieties of interest (e.g. a support and a specific bindingmember). In some cases, non-covalent linking may involve specificbinding between two moieties of interest (e.g., two affinity moietiessuch as a hapten and an antibody or a biotin moiety and a streptavidin,etc.). In certain cases, non-covalent linking may involve absorption toa substrate.

The term “polypeptide” refers to a polymeric form of amino acids of anylength, including peptides that range from 2-50 amino acids in lengthand polypeptides that are greater than 50 amino acids in length. Theterms “polypeptide” and “protein” are used interchangeably herein. Theterm “polypeptide” includes polymers of coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones in which the conventionalbackbone has been replaced with non-naturally occurring or syntheticbackbones. A polypeptide may be of any convenient length, e.g., 2 ormore amino acids, such as 4 or more amino acids, 10 or more amino acids,20 or more amino acids, 50 or more amino acids, 100 or more amino acids,300 or more amino acids, such as up to 500 or 1000 or more amino acids.“Peptides” may be 2 or more amino acids, such as 4 or more amino acids,10 or more amino acids, 20 or more amino acids, such as up to 50 aminoacids. In some embodiments, peptides are between 5 and 30 amino acids inlength.

As used herein the term “isolated,” refers to an moiety of interest thatis at least 60% free, at least 75% free, at least 90% free, at least 95%free, at least 98% free, and even at least to 99% free from othercomponents with which the moiety is associated with prior topurification.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have 5 or more, such as 6 or more, 7 or more, 8 or more, 9 or more,10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more,70 or more, 80 or more, 90 or more, 100 or more, 300 or more, 1000 ormore, 3000 or more, 10,000 or more, 100,000 or more members.

Numeric ranges are inclusive of the numbers defining the range.

The term “specific binding” refers to the ability of a capture agent (ora first member of a specific binding pair) to preferentially bind to aparticular analyte (or a second member of a specific binding pair) thatis present, e.g., in a homogeneous mixture of different analytes. Insome instances, a specific binding interaction will discriminate betweendesirable and undesirable analytes in a sample with a specificity of10-fold or more for a desirable analyte over an undesirable analytes,such as 100-fold or more, or 1000-fold or more. In some cases, theaffinity between a capture agent and analyte when they are specificallybound in a capture agent/analyte complex is at least 10⁻⁸ M, at least10⁻⁹ M, such as up to 10⁻¹⁰ M.

“Affinity” refers to the strength of binding, increased binding affinitybeing correlated with a lower Kd.

The methods described herein include multiple steps. Each step may beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step may be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step may be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

As used herein, the terms “evaluating”, “determining,” “measuring,” and“assessing,” and “assaying” are used interchangeably and include bothquantitative and qualitative determinations.

The term “separating”, as used herein, refers to physical separation oftwo elements (e.g., by size or affinity, etc.) as well as degradation ofone element, leaving the other intact.

The term “linker” or “linkage” refers to a linking moiety that connectstwo groups and has a backbone of 100 atoms or less in length. A linkeror linkage may be a covalent bond that connects two groups or a chain ofbetween 1 and 100 atoms in length, for example a chain of 1, 2, 3, 4, 5,6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, where thelinker may be linear, branched, cyclic or a single atom. In some cases,the linker is a branching linker that refers to a linking moiety thatconnects three or more groups. In certain cases, one, two, three, fouror five or more carbon atoms of a linker backbone may be optionallysubstituted with a sulfur, nitrogen or oxygen heteroatom. In some cases,the linker backbone includes a linking functional group, such as anether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate,urea, thiourea, ester, thioester or imine. The bonds between backboneatoms may be saturated or unsaturated, and in some cases not more thanone, two, or three unsaturated bonds are present in a linker backbone.The linker may include one or more substituent groups, for example withan alkyl, aryl or alkenyl group. A linker may include, withoutlimitations, polyethylene glycol; ethers, thioethers, tertiary amines,alkyls, which may be straight or branched, e.g., methyl, ethyl,n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), and the like. The linker backbone mayinclude a cyclic group, for example, an aryl, a heterocycle or acycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of thecyclic group are included in the backbone. A linker may be cleavable ornon-cleavable.

The terms “polyethylene oxide”, “PEO”, “polyethylene glycol” and “PEG”are used interchangeably and refer to a polymeric group including achain described by the formula —(CH₂—CH₂—O—)_(n)— or a derivativethereof. In some embodiments, “n” is 5000 or less, such as 1000 or less,500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 orless, 20 or less, 15 or less, such as 3 to 15, or 10 to 15. It isunderstood that the PEG polymeric group may be of any convenient lengthand may include a variety of terminal groups and/or further substituentgroups, including but not limited to, alkyl, aryl, hydroxyl, amino,acyl, acyloxy, and amido terminal and/or substituent groups. PEG groupsthat may be adapted for use in the subject multichromophores includethose PEGs described by S. Zalipsky in “Functionalized poly(ethyleneglycol) for preparation of biologically relevant conjugates”,Bioconjugate Chemistry 1995, 6 (2), 150-165; and by Zhu et al in“Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”,Chem. Rev., 2012, 112 (8), pp 4687-4735.

The term “alkyl” by itself or as part of another substituent refers to asaturated branched or straight-chain monovalent hydrocarbon radicalderived by the removal of one hydrogen atom from a single carbon atom ofa parent alkane. Alkyl groups of interest include, but are not limitedto, methyl; ethyl, propyls such as propan-1-yl or propan-2-yl; andbutyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl or2-methyl-propan-2-yl. In some embodiments, an alkyl group includes from1 to 20 carbon atoms. In some embodiments, an alkyl group includes from1 to 10 carbon atoms. In certain embodiments, a lower alkyl groupincludes from 1 to 6 carbon atoms, such as from 1 to 4 carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂−).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO— substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Amino” refers to the group —NH₂. The term “substituted amino” refers tothe group —NRR where each R is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, cycloalkyl,substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl,substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl,heteroaryl, and heterocyclyl provided that at least one R is nothydrogen.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of an aromatic ring system. Arylgroups of interest include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, 5-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like. In certain embodiments, an aryl groupincludes from 6 to 20 carbon atoms. In certain embodiments, an arylgroup includes from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

“Substituted aryl”, unless otherwise constrained by the definition forthe aryl substituent, refers to an aryl group substituted with from 1 to5 substituents, or from 1 to 3 substituents, selected from acyloxy,hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, substituted alkyl, substituted alkoxy, substitutedalkenyl, substituted alkynyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro,heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy,oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl and trihalomethyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a heteroaromatic ring system. Heteroarylgroups of interest include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene,triazole, xanthene, benzodioxole and the like. In certain embodiments,the heteroaryl group is from 5-20 membered heteroaryl. In certainembodiments, the heteroaryl group is from 5-10 membered heteroaryl. Incertain embodiments, heteroaryl groups are those derived from thiophene,pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline,imidazole, oxazole and pyrazine.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In certainembodiments, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂— moieties.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

“Substituted heteroaryl”, unless otherwise constrained by the definitionfor the substituent, refers to an heteroaryl group substituted with from1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy,hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, substituted alkyl, substituted alkoxy, substitutedalkenyl, substituted alkynyl, substituted cycloalkyl, substitutedcycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro,heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy,oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy,thioheteroaryloxy, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl and trihalomethyl.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl andsubstituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Substituents of interest include, but are not limited to, alkylenedioxy(such as methylenedioxy), -M, —R⁶⁰, —O⁻, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O)₂, —P(O)(OR⁶⁰)(O),—O P(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹,—C(O)O⁻, —C(S)OR⁶⁰, —NR⁶²C(O) NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹,—NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹,R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certainembodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O)₂O—, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O), —OP(O)(OR⁶⁰)(OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O—, —NR⁶²C(O)NR⁶⁰R⁶¹.In certain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O),—OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻. Incertain embodiments, substituents include -M, —R⁶⁰, ═O, —OR⁶⁰, —SR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰,—C(O)O R⁶⁰, —C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above. Forexample, a substituted group may bear a methylenedioxy substituent orone, two, or three substituents selected from a halogen atom, a(1-4C)alkyl group and a (1-4C)alkoxy group. When the group beingsubstituted is an aryl or heteroaryl group, the substituent(s) (e.g., asdescribed herein) may be referred to as “aryl substituent(s)”.

“Sulfonylamino” refers to the group —NR²¹SO₂R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ andR²² are optionally joined together with the atoms bound thereto to forma heterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

As summarized above, water soluble light harvesting multichromophoreshaving a plurality of pendant chromophore groups are provided. The lightharvesting multichromophore has a polymeric backbone includingnon-conjugated repeat units and a plurality of pendant donor chromophoregroups each independently linked to a non-conjugated repeat unit of thepolymeric backbone. A pendant chromophore group can be a BODIPY groupsubstituted with one or more water soluble groups. Polymeric tandem dyesbased on the subject multichromophores are also provided that furtherinclude an acceptor fluorophore linked to a non-conjugated repeat unitof the polymeric backbone and configured in energy-receiving proximityto at least one pendant donor chromophore group of the light harvestingmultichromophore. Also provided are labelled specific binding membersthat include the subject polymeric tandem dyes. Methods of evaluating asample for the presence of a target analyte and methods of labelling atarget molecule in which the subject polymeric tandem dyes find use arealso provided. Systems and kits for practicing the subject methods arealso provided.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 U.S.C.§ 112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 U.S.C. § 112 areto be accorded full statutory equivalents under 35 U.S.C. § 112.

In further describing the subject invention, light harvestingmultichromophores and related polymeric tandem dyes including anacceptor fluorophore are described first in greater detail. Next,labelled specific binding members which include the subject polymerictandem dyes are described. Then, methods of interest in which thesubject polymeric tandem dyes find use are reviewed. Systems and kitsthat may be used in practicing methods of the present disclosure arealso described.

Light Harvesting Multichromophores

As summarized above, the present disclosure includes a light harvestingmultichromophore having a modular scaffold to which pendantlight-absorbing chromophore groups are attached. The term “pendantgroup” refers to a sidechain group that is connected to the backbone ofthe modular scaffold but which is not part of the backbone. In contrastto the light absorbing co-monomers of conjugated polymer dyes, thependant light-absorbing chromophore groups of the subjectmultichromophores are not pi-conjugated to each other and do not form adelocalized pi-electron system. Rather, the modular scaffold of thesubject multichromophores provides for the configuration of a pluralityof light-absorbing chromophore groups in a compact area sufficient forefficient energy transfer between the chromophores (see e.g., FIG. 6A),and when present, to an acceptor fluorophore (see e.g., FIG. 6B). Takentogether this configuration of pendant light-absorbing chromophoregroups forms a light harvesting multichromophore having an absorptionwavelength (e.g., as described herein) at which the optically activechromophore groups absorb light to form an excited state. As such, thelight-absorbing chromophore groups are configured in energy-receivingproximity to each other and are capable of donating energy to anacceptor fluorophore when present.

The terms “light harvesting multichromophore” and “polymeric dye” areused interchangeably and refer to a polymer of the present disclosurewhich has a plurality of pendant chromophore groups capable ofharvesting light with a particular absorption maximum wavelength andconverting it to emitted light at a longer emission maximum wavelength.The polymer can have a backbone which is saturated or partiallyunsaturated.

Further pendant groups such as acceptor fluorophores, secondary donorchromophores, linkers and chemoselective tags capable of biomoleculeconjugation and water solubilizing groups can also be attached to themodular scaffold. In some cases, an acceptor fluorophore can beinstalled in conjunction with two types of donor chromophores (a primaryand a secondary donor chromophore) to provide for a desired fluorescentemission from the acceptor fluorophore. The number and positioning ofacceptor fluorophores relative to the configuration of pendant donorchromophores can be controlled.

A particular configuration of pendant groups can be determined andcontrolled by the arrangement of the repeat units of the underlyingmodular scaffold to which the pendant groups are attached. The subjectmultichromophores can include a plurality of water solubilizing groupsattached to the scaffold and/or the pendant groups at any convenientlocations to provide a water soluble light harvesting multichromophore.The modular scaffold can be composed of repeat units which form apolymeric backbone having sidechains groups to which the pendant groupscan be attached. The repeat units can be arranged in a variety ofconfigurations to provide for a water soluble light harvestingmultichromophore having desirable spectroscopic properties. Thedistances and arrangement between sites for covalent attachment of thependant donor chromophores and the acceptor fluorophore (when present)can be controlled to provide for desirable energy transfer processes.This can lead to desirable high light harvesting and signalamplification properties.

As depicted in FIG. 6A, the configuration of pendant donor chromophoregroups can exhibit, upon excitation with incident light, self-quenchingof fluorescence relative to an unquenched isolated donor chromophoregroup. By self-quenching is meant that 10% or more, such as 20% or more,25% or more, 30% or more, 40% or more, or 50% or more of thefluorescence relative to unquenched isolated donor chromophore groups.

The modular scaffold can be composed of a polymeric backbone ofnon-conjugated repeat units having any convenient configuration, such asa linear, branched or dendrimer configuration. The polymeric backbonecan be a linear polymer. The polymeric backbone can be branched. In someinstances, the light harvesting multichromophore includes a plurality ofpendant donor chromophore groups each independently linked to anon-conjugated repeat unit of the polymeric backbone. The configurationof pendant groups can be installed during or after synthesis of thepolymeric backbone. The incorporation of pendant groups can be withachieved with a random configuration, a block configuration, or in asequence-specific manner via stepwise synthesis, depending on theparticular method of synthesis utilized.

The term “unit” refers to a structural subunit of a polymer. The termunit is meant to include monomers, co-monomers, co-blocks, repeatingunits, and the like. A “repeating unit” or “repeat unit” is a subunit ofa polymer that is defined by the minimum number of distinct structuralfeatures that are required for the unit to be considered monomeric, suchthat when the unit is repeated n times, the resulting structuredescribes the polymer or a block thereof. In some cases, the polymer mayinclude two or more different repeating units, e.g., when the polymer isa multiblock polymer, a random arrangement of units or a definedsequence, each block may define a distinct repeating unit. It isunderstood that a variety of arrangements of repeating units or blocksare possible and that in the depicted formula of the subjectmultichromophores described herein any convenient linear arrangements ofvarious lengths can be included within the structure of the overallpolymer. It is understood that the polymer may also be represented by aformula in terms of mol % values of each unit in the polymer and thatsuch formula may represent a variety of arrangements of repeat unit,such as random or multiblock polymer or a defined sequence of residues.In some cases, a repeating unit of the polymer includes a single monomergroup. In certain instances, a repeating unit of the polymer includestwo or more monomer groups, i.e., co-monomer groups, such as two, three,four or more co-monomer groups. The term “co-monomer” or “co-monomergroup” refers to a structural unit of a polymer that may itself be partof a repeating unit of the polymer.

The light harvesting multichromophore includes a modular scaffold thathas a linear polymeric backbone of non-conjugated repeat units. Themodular scaffold can have a polymeric backbone including a randomconfiguration of non-conjugated repeat units. The modular scaffold canhave a polymeric backbone including a block or co-block configuration ofnon-conjugated repeat units. Alternatively, the modular scaffold canhave a polymeric backbone including a particular defined sequence ofnon-conjugated repeat units, e.g., amino acid residues of a polypeptidesequence. These configurations can be characterized by polymericsegments of repeat units (e.g., as described herein), which segments canthemselves be repeated throughout the modular scaffold.

By “non-conjugated” is meant that at least a portion of the repeat unitincludes a saturated backbone group (e.g., a group having two or moreconsecutive single covalent bonds) which precludes pi conjugation or anextended delocalized electronic structure along the polymeric backbonefrom one repeat unit to the next. It is understood that even though onerepeat unit may not be conjugated to an adjacent repeat unit, such arepeat unit may include one or more isolated unsaturated groupsincluding an unsaturated bond (e.g., of an alkenylene group or analkynylene group) and/or an aryl or heteroaryl group, which groups canbe a part of the backbone. In some cases, each repeat unit of thepolymeric backbone includes one sidechain including a linked pendantgroup or a chemoselective tag for linking to a pendant group.

In some instances, the multichromophore includes a segment of theformula (I):

wherein:

each M¹ and M² is independently an unsaturated co-monomer;

each S¹ and S² is independently a non-conjugated spacer unit;

each D¹ is independently a pendant light absorbing chromophore (e.g., asdescribed herein) linked to M¹;

each Z¹ is independently a chemoselective tag linked to M²;

x is 75 mol % or more; and

y is 25 mol % or less, where * is a connection to the polymeric backboneof the multichromophore.

In some cases of formula (I), M¹ and S¹ form a first repeat unit (M¹-S¹)and M² and S² form a second repeat unit (M²-S²) of the polymericbackbone. The first and second repeat units can be arranged in a randomor co-block configuration. In the first repeat units, D¹ can be linkedto M¹ via conjugation of a first chemoselective tag to a D¹ precursor.In the second repeat units, the Z¹ groups can be further conjugated to amolecule of interest via a second chemoselective tag (Z²) to install apendant group, such as a second light absorbing chromophore, an acceptorfluorophore or a linked biomolecule (e.g., as described herein). Incertain cases of formula (I), the D¹ pendant groups of the first repeatunits include two or more (e.g., two or three) distinct types of pendantlight harvesting chromophores that together provide a light harvestingmultichromophore system. In certain instances of formula (I), the D¹pendant groups of the first repeat units are all the same.

In some instances of formula (I), x is 80 mol % or more, such as 85 mol% or more, 90 mol % or more, 95 mol % or more, 96 mol % or more, 97 mol% or more, 98 mol % or more, or 99 mol % or more. In some instances offormula (I), y is 20 mol % or less, such as 15 mol % or less, 10 mol %or less, 5 mol % or less, 4 mol % or less, 3 mol % or less, 2 mol % orless, 1 mol % or less.

Any convenient unsaturated co-monomers can be utilized as M¹ and M²groups in the subject multichromophores, e.g., of formula (I). Byunsaturated co-monomer is meant a co-monomer having at least oneunsaturated covalent bond in the polymeric backbone. Unsaturatedco-monomers of interest include, but are not limited to, aryl orheteroaryl co-monomers, alkynyl co-monomers (e.g., ethynylene) orsegments and alkenyl co-monomers (e.g., vinylene) or segments. Aryl orheteroaryl co-monomers of interest which find use in themultichromophores (e.g., of formula (I)) include, but are not limitedto, phenyl co-monomers, biphenyl co-monomers, benzooxazole co-monomers,benzothiazole co-monomers, poly-phenylene co-monomers, and fusedtricyclic co-monomers, such as fluorene co-monomers, carbazoleco-monomers, silole co-monomers and bridged biphenyl co-monomers. Thearyl or heteroaryl co-monomers may be optionally further substituted,e.g., as described herein. In some cases of formula (I), each M¹ and M²independently includes one or more groups selected from fluorene,carbazole, silole, biphenylene and phenylene.

The light harvesting multichromophore of formula (I) includes apolymeric backbone of non-conjugated repeat units where each S¹ and S²is independently a saturated spacer unit that precludes pi-conjugationfrom between adjacent M¹ and/or M² co-monomers. In some cases, S¹ and S²are independently selected from a divalent polyethylene glycol (PEG) anda divalent modified PEG group. By divalent is meant a PEG or modifiedPEG linker that connects two adjacent co-monomers. In certain instances,the PEG or modified PEG includes 3 to 100 polyethylene glycol units,such as 6 to 100 units (e.g., PEG₆ to PEG₁₀₀).

In some instances, the multichromophore includes a segment of theformula (II):

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹, SM²and SM³ co-monomers that are each independently a non-conjugatedco-monomer;

each D¹ is independently a pendant light absorbing chromophore linked toSM¹;

each Z¹ is independently a chemoselective tag linked to SM²;

each Z² is an optional sidechain group linked to SM³;

x is 50 mol % or more; and

y+z is 50 mol % or less, where * is a connection to the polymericbackbone of the multichromophore.

Z² can be absent or any convenient sidechain group, such as a lightabsorbing chromophore, a chemoselective tag, a linker, a linkedbiomolecule, a acceptor fluorophore, a WSG, etc. In certain cases offormula (II), SM³ is a spacer co-monomer where Z² is absent. In certaininstances of formula (II), SM³ is a co-monomer including a Z² group thatis a second pendant light absorbing chromophore, where each D¹ and eachZ² together provide a light harvesting multichromophore system. In somecases, SM³ is a co-monomer including a second chemoselective tag (Z²),e.g., a protected functional group or a tag that is orthogonal to Z¹that provides for the selective installation of a moiety of interest.

In certain cases of formula (II), x is 60 mol % or more, such as 65 mol% or more, 70 mol % or more, 75 mol % or more, 80 mol % or more, 85 mol% or more, 90 mol % or more, 95 mol % or more, or even more. In certaininstances of formula (II), y+z is 40 mol % or less, such as 30 mol % orless, 25 mol % or less, 20 mol % or less, 15 mol % or less, 10 mol % orless, 5 mol % or less, or even less. In certain instances of formula(II), y is at least 1 mol % and 25 mol % or less, such as 20 mol % orless, 15 mol % or less, 10 mol % or less, 5 mol % or less, or even less.In certain instances of formula (II), z is at least 1 mol % and 10 mol %or less, such as 5 mol % or less, or even less.

In certain instances of formula (II), SM³ is absent, i.e., z is 0 mol %.As such, the multichromophore can include a segment of the formula(III):

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹ andSM² co-monomers that are each independently a non-conjugated co-monomer;

each D¹ is independently a pendant light absorbing chromophore linked toSM¹;

each Z¹ is independently a chemoselective tag linked to SM²;

x is 75 mol % or more; and

y is 25 mol % or less, where * is a connection to the polymeric backboneof the multichromophore.

In certain embodiments of formula (III), SM¹ and SM² are eachindependently a saturated non-conjugated co-monomer, e.g., a co-monomerproviding only single covalent C—C bonds. In some embodiments of formula(III), SM¹ and SM² are each independently a partially saturatednon-conjugated co-monomer, e.g., a co-monomer providing an isolateddouble C═C covalent bond in a backbone of saturated covalent bonds. Thefirst and second repeat units (SM¹ and SM²) of formula (III) can bearranged in a random configuration, a block or co-block configuration,or in a particular sequence. In the first repeat units, D¹ can be linkedto SM¹ via conjugation of a first chemoselective tag to a D¹ precursor.In the second repeat units (SM²), the Z¹ groups can be furtherconjugated to a molecule of interest via a second chemoselective tag(Z²) to install a pendant group, such as a second light harvestingchromophore, an acceptor fluorophore or a linked biomolecule (e.g., asdescribed herein). In certain cases of formula (III), the D¹ pendantgroups of the first repeat units include two or more (e.g., two orthree) distinct types of pendant light absorbing chromophores thattogether provide a light harvesting multichromophore system. In certaininstances of formula (III), the D¹ pendant groups of the first repeatunits are all the same.

In some instances of formula (III), x is 80 mol % or more, such as 85mol % or more, 90 mol % or more, 95 mol % or more, 96 mol % or more, 97mol % or more, 98 mol % or more, or 99 mol % or more. In some instancesof formula (III), y is 20 mol % or less, such as 15 mol % or less, 10mol % or less, 5 mol % or less, 4 mol % or less, 3 mol % or less, 2 mol% or less, 1 mol % or less.

Any convenient co-monomers can be utilized to provide a polymericbackbone in the multichromophore of formula (II) or (III). Co-monomersof interest which find use in preparing fully saturated or partiallysaturated polymeric backbones include, but are not limited to,co-monomers derived from an acrylate, a methacrylate, an acrylamide, apolystyrene, a ROMP (ring-opening metathesis polymerization) monomer, anADMET (acyclic diene metathesis) monomer, a cyclic carbonate, monomersderived from polyethylene glycol and monomers derived frompolyethylenimine. The co-monomers can be optionally substituted, e.g.,with a chemoselective tag. Co-monomers can be polymerized or linkedusing any convenient chemistries, including but not limited, alkenepolymerization, ring-opening polymerization, radical polymerization andClick chemistry or conjugations between compatible chemoselectivefunctional groups or tags. ADMET monomers of interest include, but arenot limited to those described by Mutlu et al. (“Acyclic dienemetathesis: a versatile tool for the construction of defined polymerarchitectures”, Chem. Soc. Rev., 2011, 40, 1404-1445). In someinstances, SM¹, SM² and/or SM³ has the following formula:

wherein:

R²¹ is -L¹-D¹ or L²-Z¹, where D¹ is a pendant donor chromophore, Z¹ is achemoselective tag and L¹ and L² are optional linkers;

n and m are independently an integer from 1 to 6 (e.g., 1 or 2); and

* is a connection to the polymeric backbone of the multichromophore.

ROMP monomers of interest include, but are not limited to, thosedescribed by Song et al. (“Scope of the Ring-Opening MetathesisPolymerization (ROMP) Reaction of 1-Substituted Cyclobutenes”, J. Am.Chem. Soc., 2010, 132 (30), pp 10513-10520). In some instances, SM¹, SM²and/or SM³ has one of the following formulae:

wherein:

R²¹ is -L¹-D¹ or L²-Z¹, where D¹ is a pendant donor chromophore, Z¹ is achemoselective tag and L¹ and L² are optional linkers;

X is CH₂ or O; and

* is a connection to the polymeric backbone of the multichromophore.

In some instances, SM¹, SM² and/or SM³ include one of the followingformulae:

wherein:

R²¹ is -L¹-D¹ or -L²-Z¹;

D¹ is a pendant donor chromophore;

Z¹ is a chemoselective tag;

L¹ and L² are optional linkers;

X is O or NR″;

R′ is H or lower alkyl (e.g., methyl);

R″ is H, lower alkyl, substituted lower alkyl and WSG; and

* is a connection to the polymeric backbone of the multichromophore. Insome instances, the polymeric backbone includes a mixture of polystyreneand acrylate or acrylamide-derived co-monomers (e.g., as describedherein).

The multichromophore can have a hydrocarbon backbone prepared using anyconvenient polymerization methods. In some cases, the hydrocarbonbackbone is derived from acrylate, acrylamide or styrene co-monomers, ora derivative thereof. In some cases, the multichromophore is describedby formula (X):

wherein:

each D¹ is independently a pendant donor chromophore;

each Z¹ is a chemoselective tag (e.g., as described herein);

each L¹, L² and L³ is independently a linker;

a, b and c are mol % values for each co-monomer;

d represents the total polymerization or average length of the polymer(e.g., d is 2-1000, such as 2-500, 2-200, 2-100 or 2-50);

WSG is a water solubilizing group (e.g., as described herein); and

G¹ and G² are each independently selected from terminal group, polymersegment, donor chromophore group, acceptor fluorophore, linker and alinked specific binding member. In some instances of formula (X), c=0.In some instances of formula (X), a>0 and b>0. In some instances offormula (X), a is 80 mol % or more, such as 85 mol % or more, 90 mol %or more, 95 mol % or more, 96 mol % or more, 97 mol % or more, 98 mol %or more, or 99 mol % or more. In some instances of formula (X), b is 20mol % or less, such as 15 mol % or less, 10 mol % or less, 5 mol % orless, 4 mol % or less, 3 mol % or less, 2 mol % or less, 1 mol % orless. In some instances of formula (X), a is 65-95 mol %, b is 5-35 mol% and c is 0-30 mol %, where a+b+c=100%. In certain instances of formula(X), L¹-L³ includes a linkage to the backbone of the polymer selectedfrom: —COO—, —CONR″—, -Ph-, —O—, where R″ is H, lower alkyl, substitutedlower alkyl or WSG. Such linkages cam be utilized to connect D¹, Z¹ andWSG to the polymer backbone.

In certain instances of formula (X), L¹-D¹ is described by one of thefollowing:

where R⁴ is H, lower alkyl, substituted lower alkyl or WSG. In certaininstances of formula (X), the WSG is a water solubilizing group asdescribed in any one of the embodiments and structures of such groupsdescribed herein.

The multichromophore can have a backbone derived from co-monomersconnected via linkages or groups that are derived from Click chemistryconjugation reactions (e.g., as described herein). Any convenientdivalent co-monomer groups can be derivatized with terminalchemoselective tags and polymerized via conjugation of compatiblechemoselective tag. FIGS. 7A and 7B illustrate exemplary schemes forpolymerization of co-monomers having alkyne and azide chemoselectivetags via Click chemistry. In some cases, the co-monomers include one ormore ethyleneoxide or ethyleneamino groups that make up part of thebackbone of the polymer. Such groups can provide for desirable watersolubility of the resulting polymeric dye. The co-monomer can furtherinclude a trivalent unit for linking to a sidechain group such as adonor or acceptor dye or a WSG. In some cases, the co-monomer includes apropyleneoxide or propyleneamino group in the backbone which is furthersubstituted at the 2-position with a sidechain group or substituent.This group can include a linked chemoselective tag, linked donor oracceptor dye, or a linked WSG.

In some instances of formula (II), the multichromophore is of formula(XXI):

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹, SM²and SM³ co-monomers that are each linked via a group T that is theproduct of a click chemistry or chemoselective group conjugationreaction (e.g., an azide-alkyne click chemistry);

SM³ optionally comprises a linked WSG;

each D¹ is independently a pendant light absorbing chromophore linked toSM¹;

each Z¹ is independently a chemoselective tag linked to SM²;

each Z² is an optional sidechain group linked to SM³;

x is 50 mol % or more; and

y+z is 50 mol % or less, where * is a connection to the polymericbackbone of the multichromophore or a terminal group, e.g., as describedherein. In certain instances of formula (XXI), SM¹, SM² and SM³ compriserepeating units selected from ethylene oxide, ethylenamino,2-substituted propyleneoxide and 2-substituted propyleneamino. In someinstances of formula (II), each T is 1,4-substituted 1,2,3-triazole,i.e., the product of azide-alkyne click chemistry conjugation reaction.

In some instances of formula (XXII), SM¹, SM² and SM³ have the followingstructures:

wherein:

each X is independently O or NR³¹ wherein R³¹ is H, alkyl, substitutedalkyl, alkanoyl or substituted alkanoyl;

each r and s is independently 1-6 (e.g., 1, 2 or 3);

each d and e is independently 1-12 (e.g., 1-6, such as 1, 2, 3, 4, 5 or6);

t is 0 or 1;

D¹ is a pendant donor chromophore;

Z¹ is a chemoselective tag (e.g., as described herein);

WSG is a water solubilizing group (e.g., as described herein);

each L¹, L² and L³ is independently a linker; and

* is a connection to a 1,4-substituted 1,2,3-triazole (T).

It is understood that for any of the co-monomers from which thestructures of SM¹-SM³ described above are derived, either an azide or analkyne group may be utilized at the terminal of the co-monomer forlinking during polymerization. As such, the 1,4-substituted1,2,3-triazole (T) may be present in one of two possible orientations asfollows:

The terminals of the polymeric backbone can include any convenientterminal groups, such as an azide or alkyne group, linker or linkedspecific binding moiety.

Exemplary multichromophore structures and precursors thereof are shownin Example 3 of the experimental section and in the followingstructures:

wherein:

G¹ is a terminal group (e.g., as described herein);

L¹ and L² are independently a linker;

D¹ is a pendant chromophore (e.g., as described herein);

each d, e and f is independently 1-6;

n is 1-1000 (e.g., 2-1000, 2-500, 2-100 or 2-50);

each R⁴¹ is selected from alkyl, substituted alkyl and WSG; and

“Linker” is a linker including an optional chemoselective functionalgroup, e.g., for conjugation to a co-monomer or a biomolecule. In someinstances of formula (XXI), L¹-L³ comprise a linkage to the backbone ofthe polymer selected from —NHCO-alkyl.

Cyclic carbonate and protected carbonate monomers of interest which canbe adapted for use in preparing polymeric backbones of the subjectmultichromophores (see e.g., formula (IX) as described herein) includes,but are not limited to, those described by Barnes et al. inWO2013036532, Cooley et al. (J. Am. Chem. Soc., 131, 45, 1640-3, 2009),and Rothbard et al. in U.S. Pat. No. 7,169,814. Such monomers areutilized in a polymerization reaction using an initiator and a suitablefeed ratio of cyclic carbonate monomers to provide a polymeric backbone.Alternative, protected carbonate monomers can be assembled in a stepwise synthesis to provide a defined sequence. In some cases of formulae(II)-(III), the polymeric backbone has a polycarbonate backbone. Assuch, the multichromophore can have formula (IX):

wherein:

each D¹ is independently a pendant donor chromophore group;

each Z¹ is independently a chemoselective tag;

each L¹ and L² is independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from the group consisting of aterminal group, a polymer segment, a donor chromophore group, anacceptor fluorophore, a linker and a linked specific binding member.

In certain embodiments, the repeat units (SM¹, SM² and/or SM³) offormulae (II)-(III) are arranged in a defined linear sequence. Anyconvenient co-monomers which can be polymerized in a defined stepwisefashion can be utilized in constructing a multichromophore of formulae(II)-(III). Co-monomers can be derived from amino acids, peptoidmonomers, or a protected or cyclic carbonate monomer.

In some cases of formulae (II)-(III), the polymeric backbone is apolypeptide having a defined sequence of α-amino acid residues and/orβ-amino acid residues. Two types of β-amino acids and polypeptides canfind use in the polymeric backbones of subject multichromophores: thosewith the sidechain group next to the amine are calledβ3-peptides/residues and those with the sidechain group next to thecarbonyl group are called β2-peptides/residues. In certain embodiments,the multichromophore is of the formula (IV):

wherein:

each D¹ is independently a pendant light absorbing chromophore group;

each Z¹ is independently a chemoselective tag;

each L¹ and L² are independently a linker;

p₁ and q₁ are independently 0 or 1 wherein p₁+q₁≤1;

p₂ and q₂ are independently 0 or 1 wherein p₁+q₁≤1;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a light absorbing (e.g., donor) chromophore group, anacceptor fluorophore, a linker and a linked specific binding member.

In some embodiments of formula (IV), p₁ and p₂ are each 0 and q₁ and q₂are each 1 (e.g., β3-amino acid residues). In some embodiments offormula (IV), p₁ and p₂ are each 1 and q₁ and q₂ are each 0 (e.g.,β2-amino acid residues). In some cases, p₁, p₂, q₁ and q₂ are each 0 andthe multichromophore is of formula (V):

wherein:

each D¹ is independently a pendant donor chromophore group;

each Z¹ is independently a chemoselective tag;

L¹ and L² are each independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a light absorbing (e.g., donor) chromophore group, anacceptor fluorophore, a linker and a linked specific binding member. Itis understood that the multichromophores described by formula (V)include any convenient arrangements of co-monomers in a defined linearsequence, which have in total the defined mol % ratios of x and y. Insome cases, the Z¹ containing co-monomers are spaced throughout thesequence of the polymeric backbone and as such are always flanked onboth sides by one or more D1 containing co-monomers.

In certain instances of formula (V), the multichromophore includes asegment of formula (VI):

wherein:

each D¹ is independently a pendant light absorbing chromophore group;

each Z¹ is independently a chemoselective tag;

each L¹ and L² are independently a linker;

n and p are each independently an integer from 1 to 20 wherein n+p 2;and

m is 1 or 2.

In some cases of formula (VI), n and p are each independently 1 to 10such as 2 to 20, 3 to 10 or 3 to 6. In some instances of formula (VI),n+p is an integer from 2 to 20, such as 3 to 20, 4 to 20, 5 to 20, 5 to15 or 5 to 12. In certain embodiments of formula (VI), m is 1.

The subject multichromophore can include multiple segments of formula(VI) where each segment includes one isolated Z¹ containing co-monomersflanked by blocks of D¹ containing co-monomers. In some cases, themultichromophore includes two or more segments of formula (VI) locateddirected adjacent to each other to provide two isolated Z¹ containingco-monomers separated by a block of 2-20 D¹ containing co-monomers, suchas a block of 3 to 20, 4 to 20, 5 to 20, 5 to 15 or 5 to 12 D¹containing co-monomers. As such, in certain embodiments, themultichromophore includes q segments of a block copolymer and is offormula (VII):

wherein: each (n)_(q) and each (p)_(q) is independently an integer from1 to 20, wherein for each of the q segments (n)_(q)+(p)_(q)≥3; and q isan integer from 1 to 100.It is understood that an α-amino acid residue of formulae (V)-(VII)could be replaced with a β2-amino acid residue or β3-amino acid residueto provide a corresponding polypeptide product. In formulae (V)-(VII),D¹ can be linked to the amino acid sidechain linker L1 via conjugationof a first chemoselective tag to a D¹ precursor. In formulae (V)-(VII),the Z¹ groups can be further conjugated to a molecule of interest via asecond chemoselective tag (Z²) to install a pendant group, such as asecond light harvesting chromophore, an acceptor fluorophore or a linkedbiomolecule (e.g., as described herein).

Any convenient amino acids can be adapted for use to provide a polymericbackbone to which pendant groups can be covalently linked. The aminoacids can be naturally occurring or non-naturally occurring. Forexample, amino acids such as lysine, ornithine have sidechain aminogroups suitable for conjugation with an amino-reactive group such as anactivated carboxylic acid. Cysteine includes a sidechain thiol groupsuitable for conjugation with a thiol-reactive group such as a maleimideor a haloacetyl. Aspartate and glutamate have sidechain carboxylic acidgroups which can be conjugated with a nucleophilic group such as anamino group. Methods of preparing the subject multichromophoresincluding peptide synthesis methods are described herein.

In some instances, the polymeric backbone of the multichromophore hasone or more of the following polypeptide sequence segments:

XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXXXXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X)_(n)YXY(X)_(n)YX XXY(X)_(n)YXX XXXY(X)_(n)YXXX XXXXY(X)_(n)YXXXXXXXXXY(X)_(n)YXXXXX

where:

each X is a first amino acid residue having a sidechain-linked firstchemoselective tag, or a sidechain-linked pendant donor chromophore;

each Y is a second amino acid residue having a sidechain-linked secondchemoselective tag, or a sidechain-linked pendant acceptor fluorophore;and

n is an integer of 2 to 20, such as 2 to 10, 3 to 10, 4 to 10 or 5 to10, e.g., n is 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In certain embodiments, in addition to the one or more polypeptidesegments described herein, a third type of amino acid residue (Z) can beincorporated into the polymeric backbone, e.g., between two of thesegments. In certain cases, a single isolated third amino acid residueis incorporated between any two of the segments described herein, e.g.,(segment 1)-Z-(segment 2). This third amino acid residue can be a spacerresidue, or a residue having a chemoselective functional group suitablefor selective installation of an additional moiety of interest, such asa second pendant light absorbing chromophore, a chemoselective tag(e.g., a bio-orthogonal click chemistry tag), a linker, a linkedbiomolecule, a acceptor fluorophore, a WSG, etc. In certain instancesthe additional moiety of interest is incorporated into the third aminoacid residue prior to preparation.

In certain instances, the first amino acid residue (X) includes asidechain amino group, e.g., lysine or ornithine or a protected versionthereof. During SPPS of the polymeric backbone, these sidechain aminogroups can remain protected (e.g., with a Cbz or Boc protecting group),and then subsequently be orthogonally deprotected to provide forconjugation of the first residues to a pendant donor chromophore group.In certain cases, the second amino acid residue includes a sidechainthiol group, e.g., cysteine or a protected version thereof. Similarly,during SPPS of the polymeric backbone, these sidechain thiol groups canremain protected, and then subsequently be orthogonally deprotected toprovide for conjugation of the second residues to a pendant group ofinterest, e.g., an acceptor fluorophore.

In some embodiments, the polymeric backbone of the multichromophore has,or is derived from, one or more of the following polypeptide sequencesegments:

(SEQ ID NO: 1) KCKK (SEQ ID NO: 2) KKCK (SEQ ID NO: 3) KKYKK(SEQ ID NO: 4) KKKYKK (SEQ ID NO: 5) KKYKKK (SEQ ID NO: 6) KKKYKKK(SEQ ID NO: 7) KKKYKKKK (SEQ ID NO: 8) KKKKYKKK (SEQ ID NO: 9) KKKKCKKKK(SEQ ID NO: 10) KKKKKCKKKKK (SEQ ID NO: 11) KKKKKKCKKKKKK(SEQ ID NO: 12) KKKKKKKCKKKKKKK (SEQ ID NO: 13) KKKKKKKKCKKKKKKKK(SEQ ID NO: 14) KKKKKKKKKCKKKKKKKKK (SEQ ID NO: 15)KKKKCKKKKKKKKKKCKKKKK (SEQ ID NO: 16) C(K)_(n)C (SEQ ID NO: 17)KC(K)_(n)CK (SEQ ID NO: 18) KKC(K)_(n)CKK (SEQ ID NO: 19)KKKC(K)_(n)CKKK (SEQ ID NO: 20) KKKKC(K)_(n)CKKKK (SEQ ID NO: 21)KKKKKC(K)_(n)CKKKKKwherein: each K is a lysine residue, a protected lysine residue, or alysine residue covalently linked via the sidechain amino group to apendant donor chromophore group; n is an integer from 2 to 20 (such as 2to 10, 3 to 10, 4 to 10 or 5 to 10, e.g., n is 2, 3, 4, 5, 6, 7, 8, 9 or10); and C is a cysteine residue or a protected cysteine residue.

In some embodiments, the polymeric backbone of the multichromophore has,or is derived from, one or more of the following polypeptide sequencesegments:

(SEQ ID NO: 22) OCOO (SEQ ID NO: 23) OOCO (SEQ ID NO: 24) OOCOO(SEQ ID NO: 25) OOOCOO (SEQ ID NO: 26) OOCOOO (SEQ ID NO: 27) OOOCOOO(SEQ ID NO: 28) OOOCOOOO (SEQ ID NO: 29) OOOOCOOO (SEQ ID NO: 30)OOOOCOOOO (SEQ ID NO: 31) OOOOOCOOOOO (SEQ ID NO: 32) OOOOOOCOOOOOO(SEQ ID NO: 33) OOOOOOOCOOOOOOO (SEQ ID NO: 34) OOOOOOOOCOOOOOOOO(SEQ ID NO: 35) OOOOOOOOOCOOOOOOOOO (SEQ ID NO: 36)OOOOOOOOOOCOOOOOOOOOO (SEQ ID NO: 37) C(O)_(n)C (SEQ ID NO: 38)OC(O)_(n)CO (SEQ ID NO: 39) OOC(O)_(n)COO (SEQ ID NO: 40)OOOC(O)_(n)COOO (SEQ ID NO: 41) OOOOC(O)_(n)COOOO (SEQ ID NO: 42)OOOOOC(O)_(n)COOOOOwherein:

O is an ornithine residue, a protected ornithine residue, or anornithine residue covalently linked via the sidechain amino group to apendant donor chromophore group; n is an integer from 2 to 20 (such as 2to 10, 3 to 10, 4 to 10 or 5 to 10, e.g., n is 2, 3, 4, 5, 6, 7, 8, 9 or10); and C is a cysteine residue or a protected cysteine residue.

In some cases of formulae (II)-(III), the polymeric backbone is apeptoid backbone. As such, the multichromophore can have formula (VIII):

wherein

each D¹ is independently a pendant donor chromophore group;

each Z¹ is independently a chemoselective tag;

each L¹ and L² is independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a light absorbing (e.g., donor) chromophore group, anacceptor fluorophore, a linker and a linked specific binding member. Itis understood that the multichromophores described by formula (VIII) caninclude any convenient arrangements of co-monomers in a defined linearsequence, which have in total the defined mol % ratios of x and y. Insome cases, the Z¹ containing co-monomers are spaced throughout thesequence of the polymeric backbone and as such are always flanked onboth sides by one or more D1 containing co-monomers.

In some instances of formulae (IV), (V), (XIII) and (IX), x is 80 mol %or more, such as 85 mol % or more, 90 mol % or more, 95 mol % or more,96 mol % or more, 97 mol % or more, 98 mol % or more, or 99 mol % ormore. In some instances of formula (IV), (V), (XIII) and (IX), y is 20mol % or less, such as 15 mol % or less, 10 mol % or less, 5 mol % orless, 4 mol % or less, 3 mol % or less, 2 mol % or less, 1 mol % orless.

Pendant Chromophore Groups

Any convenient light absorbing chromophore groups can be adapted for usein the subject multichromophores. The terms “light absorbing chromophoregroup” and “donor chromophore group” are used interchangeably and referto a pendant group of the multichromophore capable of absorbing light ata particular absorption maximum wavelength and transferring energy to aproximate chromophore or acceptor fluorophore or converting it toemitted light at a longer emission maximum wavelength.

BODIPY Chromophore Groups

A pendant chromophore group can be a BODIPY group. In some cases offormulae (I)-(IX), each D¹ is independently a BODIPY group. In somecases, the BOPIPY group is a pendant donor chromophore group. The term“BODIPY group” refers to a pendant group of the multichromophore whichincludes a chromophore having the following boron-dipyrromethene(BODIPY) core structure:

where Q is C or N and each R is any convenient boron substituent. Insome cases, Q is C. In some instances, each R is independently selectedfrom F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyland substituted alkynyl.

The BODIPY core structure may be linked to a repeat unit of themultichromophore via any convenient positions of the core structure, viaan optional sidechain linker. The BODIPY core structure may be furtheroptionally substituted. In certain embodiments, the BODIPY group definesa sidechain group of a co-monomer which is part of a repeating unit. Anyconvenient BODIPY-containing structures may be adapted for use in thesubject multichromophores as a BODIPY group. BODIPY-containingstructures of interest include, but are not limited to, those BODIPYdyes and derivatives described by Loudet and Burgess in “BODIPY Dyes andTheir Derivatives: Syntheses and Spectroscopic Properties”, Chem. Rev.2007, 107 (11): 4891-4932, Suzuki et al. in U.S. Pat. No. 8,193,350,Ulrich et al. in U.S. Pat. No. 8,476,461 and Ulrich et al. in U.S. Pat.No. 7,897,786, the disclosures of which are herein incorporated byreference in their entirety.

A BODIPY pendant chromophore group can be described by formula (XI):

wherein:

Q is C or N;

R¹-R⁷ are each independently selected from H, alkyl, substituted alkyl,alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, water solubilizing group (WSG) and -L¹-Z¹, or

-   -   optionally any one or more pairs of substituents selected from        R⁶ and R⁷, R² and R³, R⁵ and R⁶, R³ and R⁴, R⁴ and R¹ and R⁵ and        R¹, together form a divalent radical and are cyclically linked        and together with the carbon atoms to which they are bound        provide a 5- or 6-membered fused heterocycle, carbocycle, aryl        or heteroaryl ring (e.g., a 5- or 6-membered ring comprising        carbon atoms and 0-3 heteroatoms selected from O, S and N),        which ring may be unsubstituted or further substituted with a        substituent independently selected from alkyl, substituted        alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, alkenyl, substituted        alkenyl, alkynyl, substituted alkynyl, water solubilizing group        (WSG) and -L¹-Z¹;

L¹ is a linker;

Z¹ is a non-conjugated repeat unit of the polymeric backbone; and

Y¹ and Y² are independently selected from F, OH, H, cyano, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl and WSG;

wherein one of Y¹, Y² and R¹-R⁷ is linked to a non-conjugated repeatunit of the polymeric backbone. In formula (XI), it is understood thatthe substituents Y¹, Y² and R¹-R⁷ can be selected from groups that donot inhibit the fluorescence of the BODIPY group. In certain instancesof formula (XI), Q is C. It is understood that for any of the BODPIYgroup formulae described herein, a corresponding formula may be includedwhere the atom represented by Q in formula (XI) is a nitrogen atom. Incertain embodiments of formula (XI), one or more of Y¹, Y² and R¹-R⁷includes a WSG. In certain instances of formula (XI), Y¹ and Y² eachinclude a WSG. In certain instances of formula (XI), the linker of-L¹-Z¹ includes a WSG.

In formula (XI), the substituent pairs R⁶ and R⁷ and/or R² and R³ can becyclically linked to provide a 5- or 6-membered fused ring, which ringis unsubstituted or substituted. In some cases of formula (XI), the 5-or 6-membered fused ring is an aryl or heteroaryl ring selected fromfuran, thiophene, pyrrole, oxazole, isooxazole, thiazole, isothiazole,imidazole and pyrazole. In some instances of formula (XI), the BODIPYgroup is of formula (XIIa) or (XIIb):

wherein:

Z¹¹ and Z¹² are independently the fused 5- or 6-membered fusedheterocycle, carbocycle, aryl or heteroaryl ring;

each “i” is independently 0-3; and

each R²⁰ is independently a substituent group as defined for R²-R⁷ informula (X). In certain embodiments of formulae (XIIa)-(XIIb), Z¹¹ andZ¹² are independently a 5- or 6-membered fused aryl or heteroaryl ring.In some cases, Z¹¹ and Z¹² are independently selected from furan,thiophene, pyrrole, oxazole, isooxazole, thiazole, isothiazole,imidazole and pyrazole. In some cases of formulae (XIIa)-(XIIb), Z¹¹and/or Z¹² are furan or thiophene. In certain cases of formula (XIIa),none of R¹-R⁵ are cyclically linked. In some instances of formulae(XIa)-(XIIb), Z¹¹ and Z¹² are independently selected from the followingrings:

wherein:

X is O or S;

Y is O, S or NR, wherein R is H, alkyl, substituted alkyl or asubstituent as defined for R²⁰ in formulae (XIIa)-(XIIb); and

R²¹-R²³ are independently selected from H and a substituent as definedfor R²⁰ in formulae (XIIa)-(XIIb). In certain instances of formulae(XIIa)-(XIIb), Z¹¹ and Z¹² are independently selected from the followingrings:

as defined above.In certain cases of formulae (XIIb), Z¹¹ and Z¹² are the same rings. Incertain instances, Z¹¹ and Z¹² include different rings.

In certain embodiments of formula (XIIa), the BODIPY group is of formula(XIIIa) or (XIIIb):

wherein:

X is O or S: and

R¹⁶ and R¹⁷ are substituents as defined for R⁶ and R⁷ in formula (I).

In certain cases of formula (XIIIa) or (XIIIb), none of R¹-R⁵ arecyclically linked.

In certain embodiments of formula (XI) and (XIIb), the BODIPY group isof formula (XIVa) or (XIVb):

wherein:

each X is O or S; and

R¹², R¹³, R¹⁶ and R¹⁷ and are substituents as defined for R², R³, R⁶ andR⁷ in formula (I).

In certain embodiments of formulae (XI)-(XIVb), the BOBIPY groupincludes a linked chemoselective functional group or molecule ofinterest, such as a light harvesting multichromophore (e.g., -L¹-Z¹). Insome instances, one of R¹-R⁷ includes -L¹-Z¹. In certain instances, Y¹or Y² includes -L¹-Z¹. In certain embodiments of formulae (XI)-(XIVb),R¹ is -L¹-Z¹ where L¹ is a linker and Z¹ is a chemoselective functionalgroup or a light harvesting multichromophore. In certain embodiments offormulae (XI)-(XIVb), R⁴ or R⁵ is -L¹-Z¹. In certain embodiments offormulae (XI), (XIIa) and (XIIIa)-(XIIIb), R² or R⁷ is -L¹-Z¹. Incertain embodiments of formulae (XI), (XIIa) and (XIIIa)-(XIIIb), R³ orR⁶ is -L¹-Z¹. In certain embodiments of formulae (XIIa)-(XIIb), a R²⁰substituent is -L¹-Z¹. In certain embodiments of formulae(XIIIa)-(XIIIb), R¹⁶ or R¹⁷ is -L¹-Z¹. In certain embodiments offormulae (XIVa)-(XIVb), R¹², R¹³, R¹⁶ or R¹⁷ is -L¹-Z¹.

In some cases, R¹ is -L¹-Z¹ where L¹ is a linker (e.g., as describedherein) having a backbone of 20 atoms or less in length. In someinstances of R¹, L¹ is selected from an alkyl or substituted alkyllinker, an alkoxy or substituted alkoxy linker, a PEG linker, asulfonamido-alkyl or substituted sulfonamido-alkyl linker, anamido-alkyl or substituted amido-alkyl linker and an alkyl-amido-alkylor substituted alkyl-amido-alkyl linker. The linker may be substitutedwith a WSG, such as a PEG group. In certain instances of R¹, L¹ isselected from a C₁-C₁₂ alkyl or substituted alkyl linker, a C₁-C₁₂alkoxy or substituted alkoxy linker, a C₁-C₁₂ amido-alkyl or substitutedamido-alkyl linker and a C₁-C₁₂ alkyl-amido-alkyl or substitutedalkyl-amido-alkyl linker. In certain instances of R¹, Z¹ is conjugatedto the BODIPY group via a carboxylic acid or an active ester thereof.

In certain instances of formulae (XI)-(XIVb), R¹ includes an optionallysubstituted carbocyclic or heterocyclic group linked to a repeat unit ofthe light harvesting multichromophore or to a chemoselective functionalgroup. In certain instances, R¹ is an optionally substituted aryl orheteroaryl linked to a repeat unit of the light harvestingmultichromophore, e.g., via a chemoselective functional group. Bivalentcarbocyclic or heterocycle groups of interest include, but are notlimited to, 1,4-cyclohexyl, 1,3-cyclohexyl, piperidinyl (e.g.,1,4-piperidinyl), piperazinyl (e.g., 1,4-piperazinyl), and the like.Bivalent aryl or heteroaryl groups of interest include, but are notlimited to, 1,4-phenyl, 1,3-phenyl, 2,5-pyridyl, 2,6-pyridyl,3,5-pyridyl, and the like. The bivalent carbocyclic or heterocycle groupor the bivalent aryl or heteroaryl group of R¹ may be linked to -L²-Z¹,where L² is a linking group, e.g., as described in any one of theembodiments herein.

In some embodiments of formula (XI), Q is C and R¹-R⁷ are eachindependently selected from H, alkyl, substituted alkyl, alkoxy,substituted alkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkenyl, substituted alkenyl, alkynyl and substitutedalkynyl. In certain instances of formula (XI), Y¹ and Y² are eachinclude one or more water solubilizing groups (WSGs). In some cases, Y¹and Y² are an alkynyl substituted with a WSG. In some cases, Y¹ and Y²are each an alkynyl substituted with a branched WSG. In some instancesof formula (XI), Y¹ and Y² are each —CC—CH₂)_(n)—O(CH₂CH₂O)_(m)—R,wherein n is 1 to 6, m is 2 to 50, such as 2 to 30, 2 to 20, 6 to 20, 8to 20, or 10 to 20, and R is H, alkyl or substituted alkyl (e.g.,methyl).

In some embodiments of formula (XI), the BODIPY pendant donorchromophore group can be described by formula (XIa):

wherein:

* is a point of linkage to a non-conjugated repeat unit of the polymericbackbone;

Y¹ and Y² are each alkynyl substituted with one or more WSGs. In somecases, Y¹ and Y² are each alkynyl substituted with a branched WSG. Insome instances of formula (XIa), each R¹⁰ is—CC—CH₂)_(n)—O(CH₂CH₂O)_(m)—R, wherein n is 1 to 6, m is 2 to 50, suchas 2 to 30, 2 to 20, 6 to 20, 8 to 20, or 10 to 20, and R is H, alkyl orsubstituted alkyl (e.g., methyl).

In certain instances of formula (XI), the BODIPY group is of formula(XV):

wherein:

L³ is a covalent bond, oxo (—O—), alkylene (e.g., C₁-C₆-alkylene),—O-alkylene or a substituted version thereof;

R¹¹ is as defined for R¹;

each R⁹ is an optional substituent selected from halogen, hydroxyl,cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy,aryl, substituted aryl, heteroaryl and substituted heteroaryl; and t is0-4. In certain cases of formula (XV), L³ is a covalent bond. In somecases of formula (XV), L³ is oxo. In certain cases of formula (XV), L³is a covalent bond.

In certain instances of formula (XV), the BODIPY group is of formula(XVa):

wherein: R¹¹ is as defined for R¹; each R⁹ is an optional substituentselected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl,alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl andsubstituted heteroaryl; and t is 0-4. In some cases of formulae(XV)-(XVa), R¹¹ is L¹-Z¹ (e.g., as described herein). In some cases offormulae (XV)-(XVa), R¹¹ includes a sulfonamido-alkyl or substitutedsulfonamido-alkyl linker, an amido-alkyl or substituted amido-alkyllinker or an alkyl-amido-alkyl or substituted alkyl-amido-alkyl linker.In certain instances of formulae (XV)-(XVa), R², R⁴, R⁵ and R⁷ are eachindependently H, alkyl or substituted alkyl. In certain instances offormulae (XV)-(XVa), R³ and R⁶ are each independently H, alkyl orsubstituted alkyl. In some cases, R³ and R⁶ are each H. In certaininstances of formulae (XV)-(XVa), R², R⁴, R⁵ and R⁷ are eachindependently C₁-C₆alkyl or substituted C₁-C₆alkyl.

In certain instances of formula (XV), the BODIPY group is of formula(XVIa) or (XVIb):

wherein:

* is a point of linkage to a repeat unit of the light harvestingmultichromophore or a chemoselective functional group; and

Y¹ and Y² are independently selected from the group consisting of F, OH,H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, substitutedalkynyl and WSG.

In certain instances of formula (XI), the BODIPY group is of formula(XVIIa) or (XVIIb):

wherein: n is 0-12 and Z¹ is a chemoselective functional group (e.g., asdescribed herein) or linked molecule of interest. In certain cases offormulae (XVIIa)-(XVIIb), n is 1-12 or 1-6, e.g., 1, 2, 3, 4, 5 or 6. Incertain instances of formulae (XVIIa)-(XVIIb), R²-R⁴ and R⁵-R⁷ are eachindependently selected from H, C₁-C₆ alkyl and substituted C₁-C₆ alkyl.In certain instances of formulae (XVIIa)-(XVIIb), R² and R⁴ and/or R⁵and R⁷ are each independently selected from C₁-C₆ alkyl and substitutedC₁-C₆ alkyl. In certain instances of formulae (XVIIa)-(XVIIb), R³ and/orR⁶ are H. In certain instances of formulae (XVIIa)-(XVIIb), R³ and/or R⁶are alkyl or substituted alkyl.

In certain instances of formula (XIIIa), the BODIPY group is of formula(XVIII):

wherein: L¹ is a linker and Z¹ is a linked non-conjugated repeat unit ofthe polymeric backbone (e.g., as described herein). In certain instancesof formula (XVIII), the BODIPY group is of formula (XVIIIa):

wherein: L² is a linker (e.g., a linking group component of L¹); each R⁹is an optional substituent selected from halogen, hydroxyl, cyano,nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl and substituted heteroaryl; and t is 0-4.

In certain instances of formula (XIVa), the BODIPY group is of formula(XIX):

wherein: R¹² and R¹³ are substituents as defined for R² and R³ informula (XI); L¹ is a linker and Z¹ is a linked non-conjugated repeatunit of the polymeric backbone (e.g., as described herein). In certaininstances of formula (XIX), the BODIPY group is of formula (XIXa):

wherein: L² is a linker (e.g., a linking group component of L¹); each R⁹is an optional substituent selected from halogen, hydroxyl, cyano,nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl and substituted heteroaryl; and t is 0-4.

In any of the embodiments of formula (XI)-(XIXa) described herein, theBODIPY group can include a particular -L¹-Z¹ group as described in oneof the following embodiments.

L¹ can be a linker (e.g., as described herein) having a backbone of 20atoms or less in length. In some instances, L¹ is selected from an alkylor substituted alkyl linker, an alkoxy or substituted alkoxy linker, aPEG linker, a sulfonamido-alkyl or substituted sulfonamido-alkyl linker,an amido-alkyl or substituted amido-alkyl linker and analkyl-amido-alkyl or substituted alkyl-amido-alkyl linker. The linkermay be substituted with a WSG, such as a PEG group. In certaininstances, L¹ is selected from a C₁-C₁₂ alkyl or substituted alkyllinker, a C₁-C₁₂ alkoxy or substituted alkoxy linker, a C₁-C₁₂amido-alkyl or substituted amido-alkyl linker and a C₁-C₁₂alkyl-amido-alkyl or substituted alkyl-amido-alkyl linker. The linker Lcan include several linked components, such as one or linking groupsindependently selected from lower alkylene, substituted lower alkylene,alkenylene (—CH═CH—), substituted alkenylene, alkynylene (—CC—),substituted or unsubstituted amido (e.g., —NRCO— or —CONR—, where R IsH, alkyl or substituted alkyl), substituted or unsubstituted sulfonamido(e.g., —NRSO₂— or —SO₂NR—, where R Is H, alkyl or substituted alkyl),oxo (—O—), thio (—S—), ethylene glycol (—OCH₂CH₂O—), polyethylene glycol(e.g., —(CH₂CH₂O)_(n)— where n is 2-20, such as 2-10 or 2-6, or 2, 3, 4,5 or 6), arylene, substituted arylene, heteroarylene, substitutedheteroarylene, alpha-amino acid residue, beta-amino acid residue, andthe like.

The linked non-conjugated repeat unit of the polymeric backbone (Z¹) canbe conjugated to the BODIPY group via any convenient chemoselectivefunctional group, e.g., a functional group suitable for conjugation to amolecule of interest having a compatible functional group.Chemoselective functional groups of interest which find use in linkedthe subject BODIPY groups to the polymeric backbone include, but are notlimited to, amine groups (e.g., —NH₂), carboxylic acid (—CO₂H), activeester (e.g., NHS or sulfo-NHS ester), thiol, maleimide, iodoacetamide,hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, alkyne,tetrazine, alkene, phosphine and epoxide. It is understood that in somecases, the chemoselective functional group used to link a BODIPY groupis a synthetic precursor or protected version of the functional group ofinterest, which may be converted to a reactive functional group capableof conjugation the polymeric backbone. For example, a carboxylic acid isa chemoselective functional group which can be coupled with an aminegroup on a molecule of interest. The carboxylic acid may be converted toan active ester that couples with the amine group, either in situ orprior to coupling.

In some embodiments of formulae (XI)-(XIXa), L¹ includes an optionallysubstituted carbocyclic or heterocyclic group linked to a non-conjugatedrepeat unit of the polymeric backbone. In certain instances, L¹ is anoptionally substituted aryl or heteroaryl. Bivalent carbocyclic orheterocycle groups of interest include, but are not limited to,1,4-cyclohexyl, 1,3-cyclohexyl, piperidinyl (e.g., 1,4-piperidinyl),piperazinyl (e.g., 1,4-piperazinyl), and the like. Bivalent aryl orheteroaryl groups of interest include, but are not limited to,1,4-phenyl, 1,3-phenyl, 2,5-pyridyl, 2,6-pyridyl, 3,5-pyridyl, and thelike. The bivalent carbocyclic or heterocycle group or the bivalent arylor heteroaryl group of L¹ may be linked to -L²-Z¹, where L² is a linkinggroup, e.g., as described in any one of the embodiments herein. In someembodiments of formulae (I)-(IXa), -L¹-Z¹ is described by one of thefollowing structures:

wherein:

R¹¹ is L²-Z¹;

L² is a linker and Z¹ is a non-conjugated repeat unit of the polymericbackbone;

t is 0-4; and each R⁹ is independently selected from alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halogen, sulfonic acid and watersolubilizing group (WSG). In certain embodiments, -L¹-Z¹ is described byone of the following structures:

wherein:

L² is a linker and Z¹ is a non-conjugated repeat unit of the polymericbackbone;

t is 0-4; and each R⁹ is independently selected from alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halogen, sulfonic acid and watersolubilizing group (WSG). In certain embodiments, -L¹-Z¹ is described byone of the following structures:

wherein:

L² is a linker and Z¹ is a non-conjugated repeat unit of the polymericbackbone;

R³² is H, alkyl, substituted alkyl, and water solubilizing group (WSG);

L³ is a linker selected from alkylene (e.g., C₁-C₆-alkylene),—O-alkylene and substituted versions thereof;

t is 0-4; and each R⁹ is independently selected from alkyl, substitutedalkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halogen, sulfonic acid and watersolubilizing group (WSG).

In any of the embodiments of formulae (XI)-(XIXa) described herein, theBODIPY group can include particular Y¹ and Y² groups as described in oneof the following embodiments. In some cases of formulae (XI)-(XIXa), Y¹includes a water solubilizing groups (WSG). In certain cases, Y¹ isalkynyl substituted with WSG. Y² can be the same as Y¹ or different. Insome cases of formulae (XI)-(XIXa), Y² is selected from F, OH, H, cyano,alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl andsubstituted alkynyl. In some instances, Y² is selected from F, CN,phenyl and substituted phenyl.

In some cases of formulae (XI)-(XIXa), Y¹ and Y² each comprises a watersolubilizing group (WSG). In certain instances, Y¹ and/or Y² is alkynylsubstituted with a WSG (e.g., as described herein). In certaininstances, Y¹ and/or Y² is alkynyl substituted with polyethylene glycol(PEG) or modified (PEG). In certain cases of any of the embodiments offormulae (I)-(IXa), Y¹ and/or Y² is of the formula:

wherein:

s is 1 to 12;

q is 0 to 50; and

R²¹ is H, alkyl or substituted alkyl. In certain cases, s is 1 to 6,such as 1, 2 or 3. In some instances, q is 1 to 50, 1 to 30, 2 to 30, 4to 30, 6 to 30, 8 to 30, 10 to 30, 10 to 20 or 11 to 16. In certaincases, q is 10 to 50, such as 10 to 30, 10 to 20 or 11 to 16.

In certain embodiments of formula (XI) and (XVIIa), the BODIPY group hasthe structure:

where Z¹ is a linked non-conjugated repeat unit of the polymericbackbone; n is 0-6; each R, R², R⁴, R⁵ and R⁷ is independently H,C₁-C₆alkyl or substituted C₁-C₆alkyl; and each q is 6-20 (e.g., 10-20).In certain embodiments of formula (XI) and (XVIIa), the BODIPY group hasone of the following structures:

In certain embodiments of formulae (XV) and (XVa), the BODIPY dye hasthe structure:

where Z¹ is a linked non-conjugated repeat unit of the polymericbackbone; n is 0-6; R, R², R⁴, R⁵ and R⁷ is independently H, C₁-C₆alkylor substituted C₁-C₆alkyl; and each q is 6-20 (e.g., 10-20). In certainembodiments of formulae (XV), (XVa) and (XVIa) or (XVIb), the BODIPY dyehas one of the following structures:

In certain embodiments of formulae (XVIII), the BODIPY group has thestructure:

where Z¹ is a linked non-conjugated repeat unit of the polymericbackbone; n is 0-6; each R, R² and R⁴ is independently H, C₁-C₆alkyl orsubstituted C₁-C₆alkyl; and each q is 6-20 (e.g., 10-20). In certainembodiments of formulae (XVIII), the BODIPY group has one of thefollowing structures:

In certain embodiments of formulae (XIX)-(XIXa), the BODIPY group hasthe structure:

where Z¹ is a linked non-conjugated repeat unit of the polymericbackbone; n is 0-6; each R and R¹³ is independently H, C₁-C₆alkyl orsubstituted C₁-C₆alkyl; and each q is 6-20 (e.g., 10-20). In certainembodiments of formulae (XIX)-(XIXa), the BODIPY group has thestructure:

In certain embodiments of formulae (XIIIa), the BODIPY group has thestructure:

In certain embodiments of formulae (XIVa), the BODIPY group has thestructure:

In certain embodiments of formulae (XIXa), the BODIPY group has one ofthe following structures:

Aryl or Heteroaryl Chromophore Groups

The light absorbing chromophore group can be an aryl or heteroarylchromophore group. Aryl or heteroaryl chromophore groups of interestwhich find use in the subject multichromophores (e.g., of formulae(I)-(IX) include, but are not limited to, phenyl, biphenyl,benzooxazole, benzothiazole, poly-phenylene, and fused tricyclic groups,such as fluorene, carbazole, silole, biphenyl and bridged biphenyl. Thearyl or heteroaryl chromophore groups may be optionally furthersubstituted, e.g., with a water solubilizing group and/or an aryl orheteroaryl substituent that imparts desirable light absorbing propertiesto the aryl or heteroaryl group. In some cases of formulae (I)-(IX),each D¹ independently includes a fused tricyclic aryl or heteroaryl. Insome cases of formulae (I)-(IX), each D¹ independently includes one ormore groups selected from fluorene, carbazole, silole, biphenyl andbridged biphenyl.

A fused tricyclic chromophore is a group including a tricyclic aromaticgroup having three fused rings in a configuration where two aryl orheteroaryl 6-membered rings are fused to a central 5 or 6-memberedcarbocyclic or heterocyclic ring. In some cases, the fused tricyclicgroup includes two benzo or pyrido rings fused to a central 5 or 6membered carbocyclic or heterocyclic ring. The fused tricyclic group canbe linked to the sidechain of a co-monomer in the polymeric backbone viaany convenient ring atoms of the fused rings. The central 5- or6-membered ring may be a carbocycle or a heterocycle, aromatic orpartially saturated, and may further include a sidechain substituent,e.g., a WSG and/or a linker to a chemoselective tag or the co-monomersidechain. A bridged biphenyl co-monomer is a fused tricyclic grouphaving a biphenyl group where the two phenyl rings are further linkedwith each other via a central 6 membered carbocyclic or heterocyclicring.

In certain instances of the multichromophore (e.g., in formulae(I)-(IX)), a pendant donor chromophore group is a fused tricyclic arylor heteroaryl having one of the following formulae:

wherein:

* is a point of linkage to a non-conjugated repeat unit of the polymericbackbone;

Y is C(R¹³)₂, —C(R¹³)₂C(R¹³)₂—, —C(R¹³)₂Si(R¹³)₂—, NR¹³, Si(R¹³)₂ or Se;

each Z is independently CH, CR or N;

each R¹³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹¹-Z¹, where L¹¹ is alinker and Z¹ is a non-conjugated repeat unit and a WSG, or wherein anytwo convenient R³ groups are optionally cyclically linked; and

each R is independently H or one or more substituents (e.g., WSGs) andwherein any two convenient R groups are optionally cyclically linked. Insome cases, one of R and R¹³ is linked to a non-conjugated repeat unitof the polymeric backbone.

In certain instances, the fused tricyclic group is described by one ofthe following structures:

wherein Y and each R are as defined above; and the fused tricyclic groupcan be linked to the non-conjugated repeat unit of the polymericbackbone via Y or R.

In certain cases, the fused tricyclic group is a fluorene where Y isC(R³)₂. In some cases, the fused tricyclic group is a carbazole where Yis NR³. In some cases, the fused tricyclic group is a silole where Y isSi(R³)₂. In some cases, the fused tricyclic group is a bridged biphenylwhere Y is —C(R³)₂C(R³)₂— or is —C(R³)₂Si(R³)₂—. In some cases, thefused tricyclic is a bridged biphenyl where Y is —CHR³CHR³—. In certaininstances of any of the fused tricyclic groups described herein, each Ris independently selected from H, halogen, alkoxy, substituted alkoxy,alkyl and substituted alkyl. In certain cases, each R is independentlyselected from H, fluoro, chloro, methoxy, substituted alkoxy, alkyl andsubstituted alkyl.

In certain embodiments of the fused tricyclic group, the group includestwo R substituent groups that are cyclically linked to provide acarbocyclic or heterocyclic ring A that is optionally furthersubstituted:

wherein Y is C(R³)₂, —C(R³)₂C(R³)₂—, —C(R³)₂Si(R³)₂—, NR³, Si(R³)₂ orSe; and each R³ is independently selected from H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹¹-Z¹, where L¹¹ is alinker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) and a WSG; each R is as defined above;and the fused tricyclic group can be linked to the non-conjugated repeatunit of the polymeric backbone via R³ or R. In certain cases, the fusedtricyclic group has the structure:

wherein R⁸-R⁹ are each independently selected from H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹¹-Z¹, where L¹¹ is alinker and Z¹ is a chemoselective tag (e.g., a tag including achemoselective functional group) and a WSG; Y and each R is as definedabove; and the fused tricyclic group can be linked to the non-conjugatedrepeat unit of the polymeric backbone via Y, R⁸-R⁸ or R. In some casesof the co-monomer, Y is C(R³)₂.Water Solubilizing Groups

The present disclosure includes water soluble light harvestingmultichromophores having pendant chromophore groups and polymeric tandemdyes including acceptor fluorophores. The multichromophore can besubstituted with a plurality of water solubilizing groups (WSG). In somecases, the WSGs are pendant groups connected directly to the modularscaffold, e.g., as sidechains of a polymeric backbone. In certain cases,the WSGs are substituent groups attached to a pendant donor chromophoreor pendant acceptor fluorophore. In some instances, each of the pendantdonor chromophore groups are substituted with one or more WSG.

As used herein, the terms “water solubilizing group”, “water solublegroup” and WSG are used interchangeably and refer to a group orsubstituent that is well solvated in aqueous environments e.g., underphysiological conditions, and which imparts improved water solubilityupon the molecule to which it is attached. A WSG can increase thesolubility of a multichromophore in a predominantly aqueous solution, ascompared to a control multichromophore which lacks the WSG. The watersolubilizing groups may be any convenient hydrophilic group that is wellsolvated in aqueous environments.

A water soluble multichromophore of the present disclosure hassolubility under aqueous conditions that makes it especially suitablefor application to a variety of biological assays. The subject watersoluble multichromophores and polymeric tandem dyes, and conjugatesthereof, can be resistant to undesirable aggregation which providesadvantageous fluorescence and spectroscopic properties in variousbiological assays. Aggregation of dyes is undesirable because it canlead to reduced fluorescent signals, e.g., via aggregation-causedquenching of dye fluorescence. The subject water-solublemultichromophores and polymeric tandem dyes can be used as fluorescentreporters for a variety of biosensors and provide signals of exceptionalbrightness with a range of options for excitation and emissionwavelength for applications such as Flow Cytometry, and imaging.

A variety of water soluble polymer groups can be adapted for use in theWSG of the subject multichromophores. Any convenient water solubilizinggroups (WSG's) may be included in the multichromophores described hereinto provide for increased water-solubility. While the increase insolubility may vary, in some instances the increase (as compared to thecompound without the WSG(s)) is 2 fold or more, e.g., 5 fold, 10 fold,25 fold, 50 fold, 100 fold or more. In some cases, the hydrophilic watersolubilizing group is charged, e.g., positively or negatively charged.In certain cases, the hydrophilic water solubilizing group is a neutralhydrophilic group. In some embodiments, the WSG is branched (e.g., asdescribed herein). In certain instances, the WSG is linear. In someembodiments, the WSG is a hydrophilic polymer, e.g., a polyethyleneglycol, a modified PEG, a peptide sequence, a peptoid, a carbohydrate,an oxazoline, a polyol, a dendron, a dendritic polyglycerol, acellulose, a chitosan, or a derivative thereof. Water solubilizinggroups of interest include, but are not limited to, carboxylate,phosphonate, phosphate, sulfonate, sulfate, sulfinate, sulfonium, ester,polyethylene glycols (PEG) and modified PEGs, hydroxyl, amine, aminoacid, ammonium, guanidinium, pyridinium, polyamine and sulfonium,polyalcohols, straight chain or cyclic saccharides, primary, secondary,tertiary, or quaternary amines and polyamines, phosphonate groups,phosphinate groups, ascorbate groups, glycols, including, polyethers,—COOM′, —SO₃M′, —PO₃M′, —NR₃ ⁺, Y′, (CH₂CH₂O)_(p)R and mixtures thereof,where Y′ can be any halogen, sulfate, sulfonate, or oxygen containinganion, p can be 1 to 500, each R can be independently H or an alkyl(such as methyl) and M′ can be a cationic counterion or hydrogen,—(CH₂CH₂O)_(yy)CH₂CH₂XR^(yy), —(CH₂CH₂O)_(yy)CH₂CH₂X—,—X(CH₂CH₂O)_(yy)CH₂CH₂—, glycol, and polyethylene glycol, wherein yy isselected from 1 to 1000, X is selected from O, S, and NR^(ZZ), andR^(ZZ) and R^(YY) are independently selected from H and C₁₋₃ alkyl. Insome cases, a WSG is (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ where each x isindependently an integer from 0-20, each y is independently an integerfrom 0 to 50. In some cases, the water solubilizing group includes anon-ionic polymer (e.g., a PEG polymer) substituted at the terminal withan ionic group (e.g., a sulfonate).

In some embodiments of the formulae, the pendant group of interestincludes a substituent selected from (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ whereeach x is independently an integer from 0-20, each y is independently aninteger from 0 to 50; and a benzyl optionally substituted with one ormore halogen, hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_(z)OCH₃ where each zis independently an integer from 0 to 50. In some instances, thesubstituent is (CH₂)₃(OCH₂CH₂)₁₁OCH₃. In some embodiments, one or moreof the substituents is a benzyl substituted with at least one WSG groups(e.g., one or two WSG groups) selected from (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃where each x is independently an integer from 0-20 and each y isindependently an integer from 0 to 50.

Multiple WSGs may be included at a single location in the subjectmultichromophores via a branching linker. In certain embodiments, thebranching linker is an aralkyl substituent, further di-substituted withwater solubilizing groups. As such, in some cases, the branching linkergroup is a substituent of the multichromophore that connects themultichromophore to two or more water solubilizing groups. In certainembodiments, the branching linker is an amino acid, e.g., a lysine aminoacid that is connected to three groups via the amino and carboxylic acidgroups. In some cases, the incorporation of multiple WSGs via branchinglinkers imparts a desirable solubility on the multichromophore. In someinstances, the WSG is a non-ionic sidechain group capable of impartingsolubility in water in excess of 50 mg/mL. In some instances, the WSG isa non-ionic sidechain group capable of imparting solubility in water inexcess of 100 mg/mL. In some embodiments, the multichromophore includessubstituent(s) selected from the group consisting of, an alkyl, anaralkyl and a heterocyclic group, each group further substituted with ainclude water solubilizing groups hydrophilic polymer group, such as apolyethylglycol (PEG) (e.g., a PEG group of 2-20 units).

Water soluble polymers of interest that can be utilized in the WSGinclude polyethylene glycol (PEG) groups or modified PEG groups.Water-soluble polymers of interest include, but are not limited to,polyalkylene oxide based polymers, such as polyethylene glycol “PEG”(See. e.g., “Poly(ethylene glycol) Chemistry: Biotechnical andBiomedical Applications”, J. M. Harris, Ed., Plenum Press, New York,N.Y. (1992); and “Poly(ethylene glycol) Chemistry and BiologicalApplications”, J. M. Harris and S. Zalipsky, Eds., ACS (1997); andInternational Patent Applications: WO 90/13540, WO 92/00748, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28937, WO 95/11924, WO 96/00080, WO 96/23794, WO 98/07713, WO98/41562, WO 98/48837, WO 99/30727, WO 99/32134, WO 99/33483, WO99/53951, WO 01/26692, WO 95/13312, WO 96/21469, WO 97/03106, WO99/45964, and U.S. Pat. Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992;5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838;5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001;5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805;5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662;5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990;5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900;5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455;5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042;6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095;6,194,580; 6,214,966).

Examples of water soluble polymers of interest include, but are notlimited to, those containing a polyalkylene oxide, polyamide alkyleneoxide, or derivatives thereof, including polyalkylene oxide andpolyamide alkylene oxide comprising an ethylene oxide repeat unit of theformula —(CH₂—CH₂—O)—. Further examples of polymers of interest includea polyamide having a molecular weight greater than 1,000 Daltons of theformula —[C(O)—X—C(O)—NH—Y—NH]n- or —[NH—Y—NH—C(O)—X—C(O)]_(n)—, where Xand Y are divalent radicals that may be the same or different and may bebranched or linear, and n is a discrete integer from 2-100, such as from2 to 50, and where either or both of X and Y comprises a biocompatible,substantially non-antigenic water-soluble repeat unit that may be linearor branched. Further examples of water-soluble repeat units comprise anethylene oxide of the formula —(CH₂—CH₂—O)— or —(O—CH₂—CH₂)—. The numberof such water-soluble repeat units can vary significantly, with thenumber of such units being from 2 to 500, 2 to 400, 2 to 300, 2 to 200,2 to 100, 6-100, for example from 2 to 50 or 6 to 50. An example of anembodiment is one in which one or both of X and Y is selected from:—((CH₂)_(n1)—(CH₂—CH₂—O)_(n2)—(CH₂)— or—((CH₂)_(n1)—(O—CH₂—CH₂)_(n2)—(CH₂)_(n-1)—), where n1 is 1 to 6, 1 to 5,1 to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2to 8, or 2 to 5. A further example of an embodiment is one in which X is—(CH₂—CH₂)—, and where Y is —(CH₂—(CH₂—CH₂—O)₃—CH₂—CH₂—CH₂)— or—(CH₂—CH₂—CH₂—(O—CH₂—CH₂)₃—CH₂)—.

The term modified polymer, such as a modified PEG, refers to watersoluble polymers that have been modified or derivatized at either orboth terminals, e.g., to include a terminal substituent (e.g., aterminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc)and/or a terminal linking functional group (e.g., an amino or carboxylicacid group suitable for attachment via amide bond formation) suitablefor attached of the polymer to the multichromophore (e.g., via abranching group). The subject water soluble polymers can be adapted toinclude any convenient linking groups. It is understood that in somecases, the water soluble polymer can include some dispersity withrespect to polymer length, depending on the method of preparation and/orpurification of the polymeric starting materials. In some instances, thewater soluble polymers are monodisperse.

The water soluble polymer can include one or more spacers or linkers.Examples of spacers or linkers include linear or branched moietiescomprising one or more repeat units employed in a water-soluble polymer,diamino and or diacid units, natural or unnatural amino acids orderivatives thereof, as well as aliphatic moieties, including alkyl,aryl, heteroalkyl, heteroaryl, alkoxy, and the like, which can contain,for example, up to 18 carbon atoms or even an additional polymer chain.

The water soluble polymer moiety, or one or more of the spacers orlinkers of the polymer moiety when present, may include polymer chainsor units that are biostable or biodegradable. For example, polymers withrepeat linkages have varying degrees of stability under physiologicalconditions depending on bond lability. Polymers with such bonds can becategorized by their relative rates of hydrolysis under physiologicalconditions based on known hydrolysis rates of low molecular weightanalogs, e.g., from less stable to more stable, e.g., polyurethanes(—NH—C(O)—O—)>polyorthoesters (—O—C((OR)(R′))—O—)>polyamides(—C(O)—NH—). Similarly, the linkage systems attaching a water-solublepolymer to a target molecule may be biostable or biodegradable, e.g.,from less stable to more stable: carbonate (—O—C(O)—O—)>ester(—C(O)—O—)>urethane (—NH—C(O)—O—)>orthoester (—O—C((OR)(R′))—O—)>amide(—C(O)—NH—). In general, it may be desirable to avoid use of a sulfatedpolysaccharide, depending on the lability of the sulfate group. Inaddition, it may be less desirable to use polycarbonates and polyesters.These bonds are provided by way of example, and are not intended tolimit the types of bonds employable in the polymer chains or linkagesystems of the water-soluble polymers useful in the WSGs disclosedherein.

In some instances, the WSG is a branched non-ionic water soluble group(WSG) that comprises a branching group linked and provides furtherlinkages to two, three or more non-ionic water soluble polymers. In someinstances, the branched non-ionic WSG has one of the following formulae:

wherein:

each B¹ and B² are independently a branching group;

each W¹ is independently a non-ionic water soluble polymer, e.g.,comprising 6 or more monomeric units;

T³ is an optional linker to the pendant group or repeat unit of themultichromophore; and

each p and q are independently 0 or 1, wherein if present, each T¹ andeach T² are independently a linker. In certain instances, each W¹ isindependently a PEG or modified PEG polymer. In certain instances, eachW¹ is independently selected from a substituted alkyl, a PEG or modifiedPEG group and a WSG. In certain instances, each W¹ is independently aPEG or modified PEG polymer of 6-30 monomeric units, such as 6-24 or10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomeric units.

In some instances, the branched non-ionic WSG has the following formula:

wherein:

each B¹ is a branching group;

each W¹ is independently a non-ionic water soluble polymer, e.g.,comprising 6 or more monomeric units;

T³ is an optional linker to the fused 6-5-6 tricyclic co-monomer; and

each p is independently 0 or 1, wherein if present, each T¹ isindependently a linker. In certain instances, each W¹ is independently aPEG or modified PEG polymer. In certain instances, each W¹ isindependently selected from a substituted alkyl, a PEG or modified PEGgroup and a WSG. In certain instances, each W¹ is independently a PEG ormodified PEG polymer of 6-30 monomeric units, such as 6-24 or 10-30,10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomeric units. In someembodiments of the branched non-ionic WSG, B¹ is a tetra-substitutedaryl group (e.g., a 1,3,4,5-phenyl).

In some embodiments of the branched non-ionic WSG (e.g., as depicted inthe formulae above), B¹ is selected from CH, N, C(═O)N, SO₂N, atri-substituted aryl group (e.g., a 1,3,5-phenyl), a tetra-substitutedaryl group, and a tri-substituted heteroaryl group. In some embodimentsof the branched non-ionic WSG, each p is 0. In some embodiments of thebranched non-ionic WSG, p is 1, and each T¹ is selected from—(CH₂)_(n)—O—, —O—(CH₂)_(n)—, —(CH₂)_(n)— and —O—, wherein n is from 1to 12, e.g., 1 to 6. In some embodiments of the branched non-ionic WSG,each T² and/or T³ is independently a C₁-C₁₂-alkyl linker, e.g., aC₁-C₆-alkyl linker, wherein one or more backbone atoms are optionallysubstituted with a heteroatom.

In some embodiments of the subject multichromophores, the pendant donorchromophore groups are substituted with one or more water solubilizinggroups (WSGs) independently selected from the following formulae:

wherein:

T⁵ is an optional linker;

each T⁶ is an linker;

R¹¹ and R are independently H, alkyl or substituted alkyl; and

each s is an integer from 1 to 100 (e.g., 6 to 100 or 6 to 50).

In certain instances, each s is independently 6 to 30, such as 6 to 24,6 to 20, 11 to 20, 12 to 20, 12 to 18 or 12 to 16. In certain instances,each s is independently 6 to 30, such as 6 to 24, 8 to 24, 10 to 24, 12to 24, 13 to 24, 14 to 24, 15 to 22 or 16 to 20. In some cases, each sis independently 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or24. In some embodiments, each s is independently 7 or more, such as 8, 9or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15or more, or even more, and in some cases, have up to 50 monomeric units,such as up to 40, up to 30 or up to 24 monomeric units. In someinstances, each s is independently 6-30 monomeric units, such as 6-24 or10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomeric units. Insome cases, each s is the same. In some embodiments of the WSG, T⁵and/or T⁶ is a C1-C12-alkyl linker, e.g., a C1-C6-alkyl linker, whereinone or more backbone atoms are optionally substituted with a heteroatom(e.g., an —O—). In some embodiments of the WSG, each R¹¹ is H. In someembodiments of the WSG, each R¹¹ is methyl.

It is understood that hydroxy-terminated PEG chains instead ofmethoxy-terminated PEG chains may be utilized in any of the WSG groupsdescribed above. In certain instances of any one of the formulaedescribed herein, one or more of the repeat units or pendant groups issubstituted with a WSG that is a dendron selected from one of thefollowing structures:

In certain instances of any one of the formulae described herein, one ormore of the repeat units or pendant groups is substituted with a WSGthat is a polyol selected from one of the following structures:

In certain instances of any one of the formulae described herein, one ormore of the repeat units or pendant groups is substituted with WSG thatis an oxazoline of the following structure:

In certain instances of any one of the formulae described herein, one ormore of the repeat units or pendant groups is substituted with a WSGthat is a peptoid selected from one of the following structures:

The water soluble group (WSG) can be capable of imparting solubility inwater in excess of 10 mg/mL to the multichromophore or polymeric tandemdye, such as in excess of 20 mg/mL, in excess of 30 mg/mL, in excess of40 mg/mL, in excess of 50 mg/mL, in excess of 60 mg/mL, in excess of 70mg/mL, in excess of 80 mg/mL, in excess of 90 mg/mL or in excess of 100mg/mL. In certain cases, the branched non-ionic water soluble group(WSG) is capable of imparting solubility in water (e.g., an aqueousbuffer) of 20 mg/mL or more to the multichromophore or polymeric tandemdye, such as 30 mg/mL or more, 40 mg/mL or more, 50 mg/mL or more, 60mg/mL or more, 70 mg/mL or more, 80 mg/mL or more, 90 mg/mL or more, 100mg/mL or more, or even more. It is understood that water solublemultichromophores may, under certain conditions, form discrete watersolvated nanoparticles in aqueous systems. In certain cases, the watersolvated nanoparticles are resistant to aggregation and find use in avariety of biological assays.

The polymeric backbone of the multichromophore may have any convenientlength. In some cases, the particular number of monomeric repeat unitsof the multichromophore may fall within the range of 2 to 500,000, suchas 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000units or segments, or such as 5 to 100,000, 10 to 100,000, 100 to100,000, 200 to 100,000, or 500 to 50,000 units or segments. In someinstances, the particular number of monomeric repeating units orsegments of the polymeric backbone of the multichromophore may fallwithin the range of 2 to 1,000, such as 2 to 500, 2 to 500, 3 to 500, 4to 500, 5 to 500, 6 to 500, 7 to 500, 8 to 500, 9 to 500, 10 to 500, 10to 400, 10 to 300, 10 to 200, 10 to 100, or 20 to 100 units or segments.

The multichromophore may be of any convenient molecular weight (MW). Insome cases, the MW of the multichromophore may be expressed as anaverage molecular weight. In some instances, the polymeric dye has anaverage molecular weight in the range of 500 to 500,000, such as from1,000 to 100,000, from 2,000 to 100,000 (e.g., from 2,000 to 10,000 orfrom 10,000 to 100,000) or even an average molecular weight in the rangeof 50,000 to 100,000. In some cases, the polymeric backbone of themultichromophore is prepared having a particular, discrete sequence ofmonomers such that the MW of the multichromophore may be expressed as anexact molecular weight. In some instances, the polymeric dye has anexact molecular weight in the range of 500 to 500,000, such as from1,000 to 100,000, from 1,000 to 50,000, from 2,000 to 50,000 (e.g., from2,000 to 10,000 or from 10,000 to 50,000) or even an average molecularweight in the range of 50,000 to 100,000.

Polymeric Tandem Dyes

As summarized above, the subject light harvesting multichromophore caninclude a polymeric backbone of non-conjugated repeat units including aplurality of pendant donor chromophore groups, which groups can each belinked to one of the repeat units. As described above, the subject watersoluble light harvesting multichromophores are capable of homo-energytransfer between pendant donor chromophores which can lead to continuousreversible energy transfer amongst equal chromophores rather thanemission from a single chromophore. As such, although themultichromophore system can itself be fluorescent, via the process ofself-quenching illustrated in FIG. 6A, the multichromophore system canhave quantum yields that are significantly lower than those observed fora single isolated chromophore.

The water soluble light harvesting multichromophore is capable oftransferring energy to a linked acceptor fluorophore. See e.g., FIG. 6B.As such, the subject polymeric tandem dyes further include a covalentlylinked acceptor signaling fluorophore in energy-receiving proximity tothe donor water solvated light harvesting multichromophore system, i.e.,in energy-receiving proximity to at least one pendant donor chromophoregroup. The terms “acceptor fluorophore” and “acceptor chromophore” areused interchangeably herein.

The acceptor signaling fluorophore can be linked to a non-conjugatedrepeat unit of the polymeric backbone as a pendant group. Excitation ofthe multichromophore donor can leads to energy transfer to, and emissionfrom, the covalently attached acceptor signaling fluorophore. The numberof repeat units of the donor water solvated light harvestingmultichromophore having linked acceptor signaling fluorophore groups mayvary, where in some instances the number ranges from 1 mol % to 50 mol %of the repeat units, such as from 1 mol % to 25 mol %, 2 mol % to 25 mol%, 3 mol % to 25 mol %, 4 mol % to 25 mol %, 5 mol % to 25 mol % or from10 mol % to 25 mol %.

Mechanisms for energy transfer between the light harvesting chromophoresof the multichromophore and from these donor chromophores to a linkedacceptor signaling fluorophore include, for example, resonant energytransfer (e.g., Förster (or fluorescence) resonance energy transfer,FRET), quantum charge exchange (Dexter energy transfer) and the like.These energy transfer mechanisms can be relatively short range; that is,close proximity of chromophores of the light harvesting multichromophoresystem to each other and/or to an acceptor fluorophore provides forefficient energy transfer.

Under conditions for efficient energy transfer, amplification of theemission from the acceptor fluorophore can occur where the emission fromthe luminescent acceptor fluorophore is more intense when the incidentlight (the “pump light”) is at a wavelength which is absorbed by, andtransferred from, the chromophores of the light harvestingmultichromophore than when the luminescent acceptor fluorophore isdirectly excited by the pump light.

By “efficient” energy transfer is meant 10% or more, such as 20% or moreor 30% or more, 40% or more, 50% or more, of the energy harvested by thedonor chromophores is transferred to the acceptor. By “amplification” ismeant that the signal from the acceptor fluorophore is 1.5× or greaterwhen excited by energy transfer from the donor light harvestingmultichromophore system as compared to direct excitation of the acceptorfluorophore with incident light of an equivalent intensity. The signalmay be measured using any convenient method. In some cases, the 1.5× orgreater signal refers to an intensity of emitted light. In certaincases, the 1.5× or greater signal refers to an increased signal to noiseratio. In certain embodiments of the polymeric tandem dye, the acceptorfluorophore emission is 1.5 fold greater or more when excited by themultichromophore as compared to direct excitation of the acceptorfluorophore with incident light, such as 2-fold or greater, 3-fold orgreater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 8-foldor greater, 10-fold or greater, 20-fold or greater, 50-fold or greater,100-fold or greater, or even greater as compared to direct excitation ofthe acceptor fluorophore with incident light.

Any convenient fluorescent dyes may be utilized in the polymeric tandemdyes as an acceptor fluorophore. The terms “fluorescent dye” and“fluorophore” are used interchangeably herein. The acceptor fluorophore(e.g., each A¹) can be a small molecule fluorophore. The acceptorfluorophore (e.g., each A¹) can be a dye molecule selected from arhodamine, a coumarin, a xanthene, a cyanine, a polymethine, a pyrene, athiazine, an acridine, a dipyrromethene borondifluoride, a napthalimide,a phycobiliprotein, a peridinum chlorophyll protein, conjugates thereof,and combinations thereof. In certain embodiments, the acceptorfluorophore (A¹) is a cyanine dye, a xanthene dye, a coumarin dye, athiazine dye or an acridine dye. In some instances, the acceptorfluorophore (A¹) is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy 3.5 anddiethylamino coumarin. Fluorescent dyes of interest include, but are notlimited to, fluorescein, 6-FAM, rhodamine, Texas Red,tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP(peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700,7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, BODIPY 650/665, BODIPY R6G, BODIPY TMR, BODIPYTR, conjugates thereof, and combinations thereof. Lanthanide chelates ofinterest include, but are not limited to, europium chelates, terbiumchelates and samarium chelates. In some embodiments, the polymerictandem dye includes a multichromophore linked to an acceptor fluorophoreselected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647 andAlexa700. In certain embodiments, the polymeric tandem dye includes amultichromophore linked to an acceptor fluorophore selected from Dyomicsdyes (such as DY 431, DY 485XL, DY 500XL, DY 530, DY 610, DY 633, DY640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800 or DY 831), BiotiumCF 555, Cy 3.5, and diethylamino coumarin.

In some instances, the acceptor fluorophore is a BODIPY group, e.g., aBODIPY group of any one of formula (XI)-(XIXa) or any embodiment thereofdescribed herein. It is understood that any convenient BODIPY groupdescribed herein having a suitable absorption and emission profile canbe configured as an acceptor fluorophore in energy receiving proximityto the donor water solvated light harvesting multichromophore system,i.e., in energy-receiving proximity to at least one compatible pendantdonor chromophore group.

In some embodiments, the acceptor fluorophore that is selected has anemission maximum wavelength in the range of 300 to 900 nm, such as 350to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm,390 to 500 nm or 400 to 500 nm, where specific examples of emissionmaxima of signaling chromophore of interest include, but are not limitedto: 395 nm±5 nm, 420 nm±5 nm, 430 nm±5 nm, 440 nm±5 nm, 450 nm±5 nm, 460nm±5 nm, 470 nm±5 nm, 480 nm±5 nm, 490 nm±5 nm, 500 nm±5 nm, 510 nm±5nm, 520 nm±5 nm, 530 nm±5 nm, 540 nm±5 nm, 550 nm±5 nm, 560 nm±5 nm, 570nm±5 nm, 580 nm±5 nm, 590 nm±5 nm, 605 nm±5 nm, 650 nm±5 nm, 680 nm±5nm, 700 nm±5 nm, 805 nm±5 nm.

The linked luminescent acceptor fluorophore emission of the polymerictandem dye can have a quantum yield of 0.03 or more, such as a quantumyield of 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 ormore, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or moreor even more. In some instances, the polymeric tandem dye has anextinction coefficient of 5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹or more, 7×10⁵ cm⁻¹M⁻¹ or more, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ ormore, such as 1×10⁶ cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶cm⁻¹M⁻¹ or more, 2.5×10⁶ cm⁻¹M⁻¹ or more, 3×10⁶ cm⁻¹M⁻¹ or more, 4×10⁶cm⁻¹M⁻¹ or more, 5×10⁶ cm⁻¹M⁻¹ or more, 6×10⁶ cm⁻¹M⁻¹ or more, 7×10⁶cm⁻¹M⁻¹ or more, or 8×10⁶ cm⁻¹M⁻¹ or more. In some embodiments, thepolymeric tandem dye has a molar extinction coefficient of 5×10⁵ M⁻¹cm⁻¹or more. In certain embodiments, the polymeric tandem dye has a molarextinction coefficient of 1×10⁶ M⁻¹cm⁻¹ or more.

The subject polymeric tandem dyes can provide for fluorescence emissionsfrom luminescent signaling chromophore dyes that are brighter than theemissions which are possible from such luminescent dyes in isolation.The linked luminescent signaling chromophore emission of the polymerictandem dye can have a brightness of 50 mM⁻¹cm⁻¹ or more, such as 60mM⁻¹cm⁻¹ or more, 70 mM⁻¹cm⁻¹ or more, 80 mM⁻¹cm⁻¹ or more, 90 mM⁻¹cm⁻¹or more, 100 mM⁻¹cm⁻¹ or more, 150 mM⁻¹cm⁻¹ or more, 200 mM⁻¹cm⁻¹ ormore, 250 mM⁻¹cm⁻¹ or more, 300 mM⁻¹cm⁻¹ or more, or even more. Incertain instances, the linked signaling chromophore emission of thepolymeric tandem dye has a brightness that is at least 5-fold greaterthan the brightness of a directly excited luminescent dye, such as atleast 10-fold greater, at least 20-fold greater, at least 30-foldgreater, at least 50-fold greater, at least 100-fold greater, at least300-fold greater, or even greater than the brightness of a directlyexcited luminescent dye.

The subject polymeric tandem dyes can provide for fluorescence emissionshaving a Stokes shift of 100 nm or more, such as 110 nm or more, 120 nmor more, 130 nm or more, 140 nm or more, or 150 nm or more. In somecases, the Stokes shift is 300 nm or less, such as 200 nm or less.

In some embodiments, a polymeric tandem dye includes a water solublelight harvesting multichromophore of any one of formulae (I)-(IX), wherethe Z¹ chemoselective tag is replaced with an acceptor fluorophore group(A¹). It is understood that any of the embodiments of the subjectmultichromophores of formulae (I)-(IX) can also be practiced for apolymeric tandem dye of the present disclosure. In certain instances ofthe formulae (I)-(IX), one or more of the Z¹ groups can be conjugated toan acceptor fluorophore precursor to provide an acceptor fluorophoregroup (A¹). As such, the polymeric tandem dye can include a segment ofthe formula (Ia):

wherein:

each M¹ and M² is independently an unsaturated co-monomer (e.g., an arylor heteroaryl co-monomer);

each S¹ and S² is independently a non-conjugated spacer unit;

each D¹ is independently a pendant donor chromophore (e.g., as describedherein) linked to M¹;

each A¹ is independently an acceptor fluorophore linked to M²;

x is 75 mol % or more; and

y is 25 mol % or less.

The first (M¹-S¹) and second repeat units (M²-S²) can be arranged in arandom or co-block configuration. In certain cases of formula (Ia), theD¹ pendant groups of the first repeat units include two or more (e.g.,two or three) distinct types of pendant light absorbing chromophoresthat together provide a light harvesting multichromophore system. Incertain instances of formula (Ia), the D¹ pendant groups of the firstrepeat units are all the same.

In some instances of formula (Ia), x is 80 mol % or more, such as 85 mol% or more, 90 mol % or more, 95 mol % or more, 96 mol % or more, 97 mol% or more, 98 mol % or more, or 99 mol % or more. In some instances offormula (Ia), y is 20 mol % or less, such as 15 mol % or less, 10 mol %or less, 5 mol % or less, 4 mol % or less, 3 mol % or less, 2 mol % orless, 1 mol % or less.

In some instances, the polymeric tandem dye includes a segment offormula (IIa):

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹, SM²and SM³ co-monomers that are each independently a non-conjugatedco-monomer;

each D¹ is independently a pendant donor chromophore linked to SM¹;

each A¹ is independently an acceptor fluorophore linked to SM²;

each Z² is an optional sidechain group linked to SM³;

x is 50 mol % or more; and

y+z is 50 mol % or less.

Z² can be absent or any convenient sidechain group, such as a lightabsorbing chromophore, a chemoselective tag, a linker, a linkedbiomolecule, a acceptor fluorophore, a WSG, etc. In certain cases offormula (IIa), SM³ is a spacer co-monomer where Z² is absent. In certaininstances of formula (IIa), SM³ is a co-monomer including a Z² groupthat is a second pendant light absorbing chromophore, where each D¹ andeach Z² together provide a light harvesting multichromophore system. Insome cases, SM³ is a co-monomer including a second chemoselective tag(Z²), e.g., a protected functional group or a tag that is orthogonal toZ¹ that provides for the selective installation of a moiety of interest.

In certain cases of formula (IIa), x is 60 mol % or more, such as 65 mol% or more, 70 mol % or more, 75 mol % or more, 80 mol % or more, 85 mol% or more, 90 mol % or more, 95 mol % or more, or even more. In certaininstances of formula (IIa), y+z is 40 mol % or less, such as 30 mol % orless, 25 mol % or less, 20 mol % or less, 15 mol % or less, 10 mol % orless, 5 mol % or less, or even less. In certain instances of formula(IIa), y is at least 1 mol % and 25 mol % or less, such as 20 mol % orless, 15 mol % or less, 10 mol % or less, 5 mol % or less, or even less.In certain instances of formula (IIa), z is at least 1 mol % and 10 mol% or less, such as 5 mol % or less, or even less.

In some instances, the polymeric tandem dye includes a segment offormula (IIIa):

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹ andSM² co-monomers that are each independently a non-conjugated co-monomer;

each D¹ is independently a pendant donor chromophore linked to SM¹;

each A¹ is independently an acceptor fluorophore linked to SM²;

x is 75 mol % or more; and

y is 25 mol % or less.

In certain embodiments of formula (IIIa), SM¹ and SM² are eachindependently a saturated non-conjugated co-monomer, e.g., a co-monomerproviding only single covalent C—C bonds. In some embodiments of formula(IIIa), SM¹ and SM² are each independently a partially saturatednon-conjugated co-monomer, e.g., a co-monomer providing an isolateddouble C═C covalent bond in a backbone of saturated covalent bonds. Thefirst and second repeat units (SM¹ and SM²) of formula (IIIa) can bearranged in a random configuration, a block or co-block configuration,or in a particular sequence. In certain cases of formula (IIIa), the D¹pendant groups of the SM¹ include two or more (e.g., two or three)distinct types of pendant light absorbing chromophores that togetherprovide a light harvesting multichromophore system. In certain instancesof formula (IIIa), the D¹ pendant groups of the first repeat units areall the same.

In some instances of formula (IIIa), x is 80 mol % or more, such as 85mol % or more, 90 mol % or more, 95 mol % or more, 96 mol % or more, 97mol % or more, 98 mol % or more, or 99 mol % or more. In some instancesof formula (IIIa), y is 20 mol % or less, such as 15 mol % or less, 10mol % or less, 5 mol % or less, 4 mol % or less, 3 mol % or less, 2 mol% or less, 1 mol % or less.

In certain instances, the polymeric tandem dye is of formula (IVa):

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor fluorophore;

each L¹ and L² are independently a linker;

p₁ and q₁ are independently 0 or 1 wherein p₁+q₁≤1;

p₂ and q₂ are independently 0 or 1 wherein p₁+q₁≤1;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a light absorbing (e.g., donor) chromophore group, anacceptor fluorophore, a linker and a linked specific binding member.

In some embodiments of formula (IVa), p₁ and p₂ are each 0 and q₁ and q₂are each 1 (e.g., β3-amino acid residues). In some embodiments offormula (IVa), p₁ and p₂ are each 1 and q₁ and q₂ are each 0 (e.g.,β2-amino acid residues). In some cases, p₁, p₂, q₁ and q₂ are each 0 andthe polymeric tandem dye is of formula (Va):

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor fluorophore;

L¹ and L² are each independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a light absorbing (e.g., donor) chromophore group, alinker and a linked specific binding member. It is understood that themultichromophores described by formula (Va) include any convenientarrangements of co-monomers in a defined linear sequence, which have intotal the defined mol % ratios of x and y. In some cases, the A¹containing co-monomers are spaced throughout the sequence of thepolymeric backbone and as such are always flanked on both sides by oneor more D1 containing co-monomers.

In certain instances of formula (Va), the polymeric tandem dye includesa segment of formula (VIa):

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor chromophore;

each L¹ and L² are independently a linker;

n and p are each independently an integer from 1 to 20 wherein n+p 2;and

m is 1 or 2.

In some cases of formula (VIa), n and p are each independently 1 to 10such as 2 to 20, 3 to 10 or 3 to 6. In some instances of formula (VIa),n+p is an integer from 2 to 20, such as 3 to 20, 4 to 20, 5 to 20, 5 to15 or 5 to 12. In certain embodiments of formula (VIa), m is 1.

The subject polymeric tandem dyes can include multiple segments offormula (VIa) where each segment includes one isolated A¹ containingco-monomers flanked by blocks of D¹ containing co-monomers. In somecases, the multichromophore includes two or more segments of formula(VIa) located directed adjacent to each other to provide two isolated A¹containing co-monomers separated by a block of 2-20 D¹ containingco-monomers, such as a block of 3 to 20, 4 to 20, 5 to 20, 5 to 15 or 5to 12 D¹ containing co-monomers. As such, in certain embodiments, thepolymeric tandem dye includes q segments of a block copolymer and is offormula (Vila):

wherein: each (n)_(q) and each (p)_(q) is independently an integer from1 to 20, wherein for each of the q segments (n)_(q)+(p)_(q)≥3; and q isan integer from 1 to 100.

In certain embodiments, the polymeric tandem dye has the formula(VIIIa):

wherein

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor chromophore;

each L¹ and L² is independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a donor chromophore group, a linker and a linkedspecific binding member.

In certain embodiments, the polymeric tandem dye has the formula (IXa):

wherein:

each D¹ is independently a pendant BODIPY donor chromophore;

each A¹ is independently an acceptor fluorophore;

each L¹ and L² is independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from the group consisting of aterminal group, a polymer segment, a donor chromophore group, anacceptor fluorophore, a linker and a linked specific binding member.

In some instances of formulae (IVa), (Va), (XIIIa) and (IXa), x is 80mol % or more, such as 85 mol % or more, 90 mol % or more, 95 mol % ormore, 96 mol % or more, 97 mol % or more, 98 mol % or more, or 99 mol %or more. In some instances of formula (IV), (V), (XIII) and (IX), y is20 mol % or less, such as 15 mol % or less, 10 mol % or less, 5 mol % orless, 4 mol % or less, 3 mol % or less, 2 mol % or less, 1 mol % orless.

In certain embodiments, the polymeric tandem dye has the formula (Xa):

wherein:

each D¹ is independently a pendant donor chromophore;

each A¹ is independently an acceptor fluorophore;

each L¹, L² and L³ is independently a linker;

a, b and c are mol % values for each co-monomer;

d represents the total polymerization or average length of the polymer(e.g., d is 2-1000, such as 2-500, 2-200, 2-100 or 2-50);

WSG is a water solubilizing group (e.g., as described herein); and

G¹ and G² are each independently selected from the group consisting of aterminal group, a polymer segment, a donor chromophore group, anacceptor fluorophore, a linker and a linked specific binding member.

In some instances of formula (Xa), c=0. In some instances of formula(Xa), a>0 and b>0. In some instances of formula (Xa), a is 80 mol % ormore, such as 85 mol % or more, 90 mol % or more, 95 mol % or more, 96mol % or more, 97 mol % or more, 98 mol % or more, or 99 mol % or more.In some instances of formula (Xa), b is 20 mol % or less, such as 15 mol% or less, 10 mol % or less, 5 mol % or less, 4 mol % or less, 3 mol %or less, 2 mol % or less, 1 mol % or less. In some instances of formula(Xa), a is 65-95 mol %, b is 5-35 mol % and c is 0-30 mol %, wherea+b+c=100%.

In certain instances of formula (Xa), each D¹ is a linked BODIPY group(e.g., as described herein). In some cases, each acceptor dye A¹ ischosen from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682,DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778,DY 782, DY 800, DY 831, iFluor dyes 350, 405, 488, 514, 532, 594, 660,680, 700, 710, 790, Tide Fluor dyes 5WS, 7WS, 8ws, ICG, BODIPY-baseddyes, Biotium CF 555, Cy 3.5 and diethylamino coumarin. In certaininstances of formula (Xa), L¹-D¹ is selected from the following:

where R⁴ is H, lower alkyl, substituted lower alkyl and WSG.

Alternatively, in some cases of formula (Xa), pairs of donor andacceptor dyes D¹ and A¹ can be chosen from the following: Dyomics dyesDY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY800, DY 831, iFluor dyes 350, 405, 488, 514, 532, 594, 660, 680, 700,710, 790, Tide Fluor dyes 5WS, 7WS, 8ws, ICG, BODIPY-based dyes, BiotiumCF 555, Cy 3.5 and diethylamino coumarin. In certain instances offormula (Xa), the WSG is a water solubilizing group as described in anyone of the embodiments and structures of such groups described herein.

In certain instances, the polymeric tandem dye is derived from amultichromophore of formula (XXI), wherein the chemoselective tag Z¹ ofSM² is conjugated to an acceptor fluorophore. As such, exemplarypolymeric tandem dye structures are shown in Example 3 of theexperimental section and in the following structures:

wherein:

G¹ is a terminal group (e.g., as described herein);

L¹ and L² are independently a linker;

D¹ is a pendant chromophore (e.g., as described herein);

A¹ is an acceptor fluorophore;

each d, e, f, g and h are independently 1-6;

each n and m is independently 1-1000 (e.g., 2-1000, 2-500, 2-100 or2-50);

each R⁴¹ is selected from alkyl, substituted alkyl and WSG; and

“Linker” is a linker including an optional chemoselective functionalgroup, e.g., for conjugation to a co-monomer or a biomolecule. Incertain instance, each D¹ is a BODIPY chromophore group (e.g., asdescribed herein). In some instances, the polymeric tandem dye derivedfrom formula (XXI) is described by the following general structure:

where each n and m is independently 1-1000 (e.g., 2-1000, 2-500, 2-100or 2-50). In some cases, the donor chromophore is a BODIPY chromophore(e.g., as described herein), and the acceptor dye is selected from DY431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701,DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800,DY 831, iFluor dyes 350, 405, 488, 514, 532, 594, 660, 680, 700, 710,790, Tide Fluor dyes 5WS, 7WS, 8ws, ICG, BODIPY-based dyes, Biotium CF555, Cy 3.5 and diethylamino coumarin.Labelled Specific Binding Members

Aspects of the present disclosure include labelled specific bindingmembers. A labelled specific binding member is a conjugate of a subjectpolymeric dye (e.g., as described herein) and a specific binding member.Any of the polymeric dyes or polymeric tandem dyes described herein maybe conjugated to a specific binding member. The specific binding memberand the polymeric dye can be conjugated (e.g., covalently linked) toeach other at any convenient locations of the two molecules, via anoptional linker. In some embodiments, the labelled specific bindingmember is aggregation resistant. As used herein, by“aggregation-resistant” is meant a labelled specific binding membercapable of forming a homogenous aqueous composition without aggregatedprecipitate at a concentration of 1 mg/ml or more in an aqueous bufferof interest, such as 2 mg/ml or more, 3 mg/ml or more, 4 mg/ml or more,5 mg/ml or more, 6 mg/ml or more, 7 mg/ml or more, 8 mg/ml or more, 9mg/ml or more, 10 mg/mL or more or even more of the labelled specificbinding member.

In certain embodiments, the labelled specific binding member comprises:a water solvated polymeric dye having a deep ultraviolet excitationspectrum and comprising a segment of π-conjugated co-monomers and aconjugation-modifying repeat unit; and a specific binding membercovalently linked to the multichromophore.

As used herein, the term “specific binding member” refers to one memberof a pair of molecules which have binding specificity for one another.One member of the pair of molecules may have an area on its surface, ora cavity, which specifically binds to an area on the surface of, or acavity in, the other member of the pair of molecules. Thus, the membersof the pair have the property of binding specifically to each other toproduce a binding complex. In some embodiments, the affinity betweenspecific binding members in a binding complex is characterized by aK_(d) (dissociation constant) of 10⁻⁶ M or less, such as 10⁻⁷ M or less,including 10⁻⁸ M or less, e.g., 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M or less, including10⁻¹⁵ M or less. In some embodiments, the specific binding membersspecifically bind with high avidity. By high avidity is meant that thebinding member specifically binds with an apparent affinitycharacterized by an apparent K_(d) of 10×10⁻⁹ M or less, such as 1×10⁻⁹M or less, 3×10⁻¹⁰ M or less, 1×10⁻¹⁰ M or less, 3×10⁻¹¹ M or less,1×10⁻¹¹ M or less, 3×10⁻¹² M or less or 1×10⁻¹² M or less.

The specific binding member can be proteinaceous. As used herein, theterm “proteinaceous” refers to a moiety that is composed of amino acidresidues. A proteinaceous moiety can be a polypeptide. In certain cases,the proteinaceous specific binding member is an antibody. In certainembodiments, the proteinaceous specific binding member is an antibodyfragment, e.g., a binding fragment of an antibody that specific binds toa polymeric dye. As used herein, the terms “antibody” and “antibodymolecule” are used interchangeably and refer to a protein consisting ofone or more polypeptides substantially encoded by all or part of therecognized immunoglobulin genes. The recognized immunoglobulin genes,for example in humans, include the kappa (k), lambda (l), and heavychain genetic loci, which together comprise the myriad variable regiongenes, and the constant region genes mu (u), delta (d), gamma (g), sigma(e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypesrespectively. An immunoglobulin light or heavy chain variable regionconsists of a “framework” region (FR) interrupted by three hypervariableregions, also called “complementarity determining regions” or “CDRs”.The extent of the framework region and CDRs have been precisely defined(see, “Sequences of Proteins of Immunological Interest,” E. Kabat etal., U.S. Department of Health and Human Services, (1991)). Thenumbering of all antibody amino acid sequences discussed herein conformsto the Kabat system. The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs. The CDRs are primarily responsible for binding to an epitopeof an antigen. The term antibody is meant to include full lengthantibodies and may refer to a natural antibody from any organism, anengineered antibody, or an antibody generated recombinantly forexperimental, therapeutic, or other purposes as further defined below.

Antibody fragments of interest include, but are not limited to, Fab,Fab′, F(ab′)₂, Fv, scFv, or other antigen-binding subsequences ofantibodies, either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA technologies. Antibodiesmay be monoclonal or polyclonal and may have other specific activitieson cells (e.g., antagonists, agonists, neutralizing, inhibitory, orstimulatory antibodies). It is understood that the antibodies may haveadditional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other antibody functions.

In certain embodiments, the specific binding member is a Fab fragment, aF(ab′)₂ fragment, a scFv, a diabody or a triabody. In certainembodiments, the specific binding member is an antibody. In some cases,the specific binding member is a murine antibody or binding fragmentthereof. In certain instances, the specific binding member is arecombinant antibody or binding fragment thereof.

In some embodiments, the labelled specific binding member includes: awater solvated light harvesting multichromophore (e.g., as describedherein); and a signaling chromophore covalently linked to themultichromophore in energy-receiving proximity therewith (e.g., asdescribed herein); and a specific binding member covalently linked tothe multichromophore. In some instances of the labelled specific bindingmember, the multichromophore of any of the formula described herein,wherein: G¹ and G² are each independently selected from the groupconsisting of a terminal group (e.g., end group), a linker and a linkedspecific binding member, wherein at least one of G¹ and G² is a linkedspecific binding member.

In certain embodiments of the formulae described herein, G¹ and/or G² isa linker, such as a linker including a functional group suitable forconjugation to a specific binding moiety. It is understood that linkerslocated at the G¹ and/or G² positions of the multichromophore may beselected so as to be orthogonal to any other linkers includingchemoselective tags (e.g., as described herein) that may be present at asidechain of the multichromophore (e.g., at Z²). In certain embodiments,an amino functional group or derivative thereof is included at G¹ and/orG² and a carboxylic acid functional group or derivative thereof isincluded at Z². In certain embodiments, a carboxylic acid functionalgroup or derivative thereof is included at G¹ and/or G² and an aminofunctional group or derivative thereof is included at Z².

In some embodiments of the formulae described herein, at least one of G¹and G² is -L³-Z⁴ where L³ is a linker (e.g., as described herein) and Z⁴is a specific binding member (e.g., as described herein). In someembodiments of formulae described herein, at least one of G¹ and G² is-L³-Z³ where L³ is a linker (e.g., as described herein) and Z³ is achemoselective tag (e.g., as described herein). Any convenientchemoselective tag and conjugation chemistries can be adapted for use inthe subject multichromophores. Chemoselective tags of interest include,but are not limited to, amine, active ester, maleimide, thiol,sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, DiersAlder cycloaddition click reagents and click chemistry, tetrazine,transcyclooctene, aldehyde, alkoxylamine, alkynes, cyclooctynes, azide,and the like. In some instances, Z³ is selected from the groupconsisting of carboxylic acid, active ester (e.g., N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS), amino, maleimide, iodoacetyl andthiol.

Biomolecules of interest include, but are not limited to, polypeptides,polynucleotides, carbohydrates, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs thereof and combinationsthereof. In certain instances, Z⁴ is an antibody. In some instances, Z⁴is an antibody fragment or binding derivative thereof. In some cases,the antibody fragment or binding derivative thereof is selected from thegroup consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, adiabody and a triabody.

Methods of Use

As summarized above, aspects of the present disclosure include methodsof evaluating a sample for the presence of a target analyte. Aspects ofthe method include contacting the sample with a polymeric dye conjugatethat specifically binds the target analyte to produce a labellingcomposition contacted sample. As used herein, the terms “polymeric dyeconjugate” and “labelled specific binding member” are usedinterchangeably. As such, the polymeric dye conjugate can include: (i) awater solvated polymeric dye (e.g., as described herein); and (ii) aspecific binding member (e.g., as described herein). In some instances,the polymeric dye conjugate further comprises a signaling chromophorecovalently linked to a multichromophore of the polymeric dye inenergy-receiving proximity therewith.

Any convenient method may be used to contact the sample with a polymericdye conjugate that specifically binds to the target analyte to producethe labelling composition contacted sample. In some instances, thesample is contacted with the polymeric dye conjugate under conditions inwhich the specific binding member specifically binds to the targetanalyte, if present. For specific binding of the specific binding memberof the conjugate with the target analyte, an appropriate solution may beused that maintains the biological activity of the components of thesample and the specific binding member. The solution may be a balancedsalt solution, e.g., normal saline, PBS, Hank's balanced salt solution,etc., conveniently supplemented with fetal calf serum, human plateletlysate or other factors, in conjunction with an acceptable buffer at lowconcentration, such as from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc. Various media are commerciallyavailable and may be used according to the nature of the target analyte,including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., in some casessupplemented with fetal calf serum or human platelet lysate. The finalcomponents of the solution may be selected depending on the componentsof the sample which are included.

The temperature at which specific binding of the specific binding memberof the conjugate to the target analyte takes place may vary, and in someinstances may range from 5° C. to 50° C., such as from 10° C. to 40° C.,15° C. to 40° C., 20° C. to 40° C., e.g., 20° C., 25° C., 30° C., 35° C.or 37° C. (e.g., as described above). In some instances, the temperatureat which specific binding takes place is selected to be compatible withthe biological activity of the specific binding member and/or the targetanalyte. In certain instances, the temperature is 25° C., 30° C., 35° C.or 37° C. In certain cases, the specific binding member is an antibodyor fragment thereof and the temperature at which specific binding takesplace is room temperature (e.g., 25° C.), 30° C., 35° C. or 37° C. Anyconvenient incubation time for specific binding may be selected to allowfor the formation of a desirable amount of binding complex, and in someinstances, may be 1 minute (min) or more, such as 2 min or more, 10 minor more, 30 min or more, 1 hour or more, 2 hours or more, or even 6hours or more.

Any convenient specific binding members may be utilized in theconjugate. Specific binding members of interest include, but are notlimited to, those agents that specifically bind cell surface proteins ofa variety of cell types, including but not limited to, stem cells, e.g.,pluripotent stem cells, hematopoietic stem cells, T cells, T regulatorcells, dendritic cells, B Cells, e.g., memory B cells, antigen specificB cells, granulocytes, leukemia cells, lymphoma cells, virus cells(e.g., HIV cells) NK cells, macrophages, monocytes, fibroblasts,epithelial cells, endothelial cells, and erythroid cells. Target cellsof interest include cells that have a convenient cell surface marker orantigen that may be captured by a convenient specific binding memberconjugate. In some embodiments, the target cell is selected from HIVcontaining cell, a Treg cell, an antigen-specific T-cell populations,tumor cells or hematopoetic progenitor cells (CD34+) from whole blood,bone marrow or cord blood. Any convenient cell surface proteins or cellmarkers may be targeted for specific binding to polymeric dye conjugatesin the subject methods. In some embodiments, the target cell includes acell surface marker selected from a cell receptor and a cell surfaceantigen. In some cases, the target cell may include a cell surfaceantigen such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25,CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34,CD1c, CD23, CD304, CD235a, T cell receptor alpha/beta, T cell receptorgamma/delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5(N-Terminal), SSEA-3, TRA-1-60 Antigen, Disialoganglioside GD2 and CD71.

Any convenient targets may be selected for evaluation utilizing thesubject methods. Targets of interest include, but are not limited to, anucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule, aprotein, such as a fusion protein, a modified protein, such as aphosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylatedprotein, or an antibody, a peptide, an aggregated biomolecule, a cell, asmall molecule, a vitamin and a drug molecule. As used herein, the term“a target protein” refers to all members of the target family, andfragments thereof. The target protein may be any protein of interest,such as a therapeutic or diagnostic target, including but not limitedto: hormones, growth factors, receptors, enzymes, cytokines,osteoinductive factors, colony stimulating factors and immunoglobulins.The term “target protein” is intended to include recombinant andsynthetic molecules, which can be prepared using any convenientrecombinant expression methods or using any convenient syntheticmethods, or purchased commercially. In some embodiments, the polymericdye conjugates include an antibody or antibody fragment. Any convenienttarget analyte that specifically binds an antibody or antibody fragmentof interest may be targeted in the subject methods.

In some embodiments, the target analyte is associated with a cell. Incertain instances, the target analyte is a cell surface marker of thecell. In certain cases, the cell surface marker is selected from thegroup consisting of a cell receptor and a cell surface antigen. In someinstances, the target analyte is an intracellular target, and the methodfurther includes lysing the cell.

In some embodiments, the sample may include a heterogeneous cellpopulation from which target cells are isolated. In some instances, thesample includes peripheral whole blood, peripheral whole blood in whicherythrocytes have been lysed prior to cell isolation, cord blood, bonemarrow, density gradient-purified peripheral blood mononuclear cells orhomogenized tissue. In some cases, the sample includes hematopoeticprogenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cordblood. In certain embodiments, the sample includes tumor cells inperipheral blood. In certain instances, the sample is a sample including(or suspected of including) viral cells (e.g., HIV).

The labelled specific binding members find use in the subject methods,e.g., for labeling a target cell, particle, target or analyte with apolymeric dye or polymeric tandem dye. For example, labelled specificbinding members find use in labeling cells to be processed (e.g.,detected, analyzed, and/or sorted) in a flow cytometer. The labelledspecific binding members may include antibodies that specifically bindto, e.g., cell surface proteins of a variety of cell types (e.g., asdescribed herein). The labelled specific binding members may be used toinvestigate a variety of biological (e.g., cellular) properties orprocesses such as cell cycle, cell proliferation, cell differentiation,DNA repair, T cell signaling, apoptosis, cell surface protein expressionand/or presentation, and so forth. Labelled specific binding members maybe used in any application that includes (or may include)antibody-mediated labeling of a cell, particle or analyte.

In some instances of the method, the labelled specific binding memberincludes a multichromophore as described herein (e.g., according to anyone of formulae (I)-(IX)). In certain cases, G¹ and G² are eachindependently selected from the group consisting of a terminal group, apolymeric segment, a linker, a chemoselective tag and a linked specificbinding member, wherein at least one of G¹ and G² is a linked specificbinding member.

Aspects of the method include assaying the labelling compositioncontacted sample for the presence of a polymeric dye conjugate-targetanalyte binding complex to evaluate whether the target analyte ispresent in the sample. Once the sample has been contacted with thepolymeric dye conjugate, any convenient methods may be utilized inassaying the labelling composition contacted sample that is produced forthe presence of a polymeric dye conjugate-target analyte bindingcomplex. The polymeric dye conjugate-target analyte binding complex isthe binding complex that is produced upon specific binding of thespecific binding member of the conjugate to the target analyte, ifpresent. Assaying the labelling composition contacted sample can includedetecting a fluorescent signal from the binding complex, if present. Insome cases, the assaying includes a separating step where the targetanalyte, if present, is separated from the sample. A variety of methodscan be utilized to separate a target analyte from a sample, e.g., viaimmobilization on a support. Assay methods of interest include, but arenot limited to, any convenient methods and assay formats where pairs ofspecific binding members such as avidin-biotin or hapten-anti-haptenantibodies find use, are of interest. Methods and assay formats ofinterest that may be adapted for use with the subject compositionsinclude, but are not limited to, flow cytometry methods, in-situhybridization methods, enzyme-linked immunosorbent assays (ELISAs),western blot analysis, magnetic cell separation assays and fluorochromepurification chromatography.

In certain embodiments, the method further includes contacting thesample with a second specific binding member that specifically binds thetarget analyte. In certain instances, the second specific binding memberis support bound. Any convenient supports may be utilized to immobilizea component of the subject methods (e.g., a second specific bindingmember). In certain instances, the support is a particle, such as amagnetic particle. In some instances, the second specific binding memberand the polymeric dye conjugate produce a sandwich complex that may beisolated and detected, if present, using any convenient methods. In someembodiments, the method further includes flow cytometrically analyzingthe polymeric dye conjugate-target analyte binding complex, i.e., afluorescently labelled target analyte. Assaying for the presence of apolymeric dye conjugate-target analyte binding complex may provide assayresults (e.g., qualitative or quantitative assay data) which can be usedto evaluate whether the target analyte is present in the sample.

Any convenient supports may be utilized in the subject methods toimmobilize any convenient component of the methods, e.g., labelledspecific binding member, target, secondary specific binding member, etc.Supports of interest include, but are not limited to: solid substrates,where the substrate can have a variety of configurations, e.g., a sheet,bead, or other structure, such as a plate with wells; beads, polymers,particle, a fibrous mesh, hydrogels, porous matrix, a pin, a microarraysurface, a chromatography support, and the like. In some instances, thesupport is selected from the group consisting of a particle, a planarsolid substrate, a fibrous mesh, a hydrogel, a porous matrix, a pin, amicroarray surface and a chromatography support. The support may beincorporated into a system that it provides for cell isolation assistedby any convenient methods, such as a manually-operated syringe, acentrifuge or an automated liquid handling system. In some cases, thesupport finds use in an automated liquid handling system for the highthroughput isolation of cells, such as a flow cytometer.

In some embodiments of the method, the separating step includes applyingan external magnetic field to immobilize a magnetic particle. Anyconvenient magnet may be used as a source of the external magnetic field(e.g., magnetic field gradient). In some cases, the external magneticfield is generated by a magnetic source, e.g. by a permanent magnet orelectromagnet. In some cases, immobilizing the magnetic particles meansthe magnetic particles accumulate near the surface closest to themagnetic field gradient source, i.e. the magnet.

The separating may further include one or more optional washing steps toremove unbound material of the sample from the support. Any convenientwashing methods may be used, e.g., washing the immobilized support witha biocompatible buffer which preserves the specific binding interactionof the polymeric dye and the specific binding member. Separation andoptional washing of unbound material of the sample from the supportprovides for an enriched population of target cells where undesiredcells and material may be removed.

In certain embodiments, the method further includes detecting thelabelled target. Detecting the labelled target may include exciting themultichromophore with one or more lasers and subsequently detectingfluorescence emission from the polymeric tandem dye using one or moreoptical detectors. Detection of the labelled target can be performedusing any convenient instruments and methods, including but not limitedto, flow cytometry, FACS systems, fluorescence microscopy; fluorescence,luminescence, ultraviolet, and/or visible light detection using a platereader; high performance liquid chromatography (HPLC); and massspectrometry. When using fluorescently labeled components in the methodsand compositions of the present disclosure, it is recognized thatdifferent types of fluorescence detection systems can be used topractice the subject methods. In some cases, high throughput screeningcan be performed, e.g., systems that use 96 well or greater microtiterplates. A variety of methods of performing assays on fluorescentmaterials can be utilized, such as those methods described in, e.g.,Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B., Resonance energy transfer microscopy,in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methodsin Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego:Academic Press (1989), pp. 219-243; Turro, N.J., Modern MolecularPhotochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc.(1978), pp. 296-361.

Fluorescence in a sample can be measured using a fluorimeter. In somecases, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescently labelled targets in the sample emit radiation which has awavelength that is different from the excitation wavelength. Collectionoptics then collect the emission from the sample. The device can includea temperature controller to maintain the sample at a specifictemperature while it is being scanned. In certain instances, amulti-axis translation stage moves a microtiter plate holding aplurality of samples in order to position different wells to be exposed.The multi-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation.

In some embodiments, the method of evaluating a sample for the presenceof a target analyte further includes detecting fluorescence in a flowcytometer. In some embodiments, the method of evaluating a sample forthe presence of a target analyte further includes imaging the labellingcomposition contacted sample using fluorescence microscopy. Fluorescencemicroscopy imaging can be used to identify a polymeric dyeconjugate-target analyte binding complex in the contacted sample toevaluate whether the target analyte is present. Microscopy methods ofinterest that find use in the subject methods include laser scanningconfocal microscopy.

Also provided are methods of labelling a target molecule. The subjectpolymeric dyes, find use in a variety of methods of labelling,separation, detection and/or analysis. In some embodiments, the methodincludes: contacting the target molecule with a polymeric dye (e.g., asdescribed herein) to produce a labelled target molecule, wherein thepolymeric dye includes a conjugation tag that covalently links to thetarget molecule. In some cases, the polymeric dye further includes asignaling chromophore covalently linked to the multichromophore of thepolymeric dye in energy-receiving proximity therewith. In some instancesof the method, the polymeric dye member includes a multichromophoreaccording to any one of formulae (I)-(IX) (e.g., as described herein),where one of G¹ and G² is a terminal group and the other of G¹ and G² isthe conjugation tag.

As used herein the term “conjugation tag” refers to a group thatincludes a chemoselective functional group (e.g., as described herein)that can covalently link with a compatible functional group of a targetmolecule, after optional activation and/or deprotection. Any convenientconjugation tags may be utilized in the subject polymeric dyes in orderto conjugate the dye to a target molecule of interest. In someembodiments, the conjugation tag includes a terminal functional groupselected from an amino, a carboxylic acid or a derivative thereof, athiol, a hydroxyl, a hydrazine, a hydrazide, a azide, an alkyne and aprotein reactive group (e.g. amino-reactive, thiol-reactive,hydroxyl-reactive, imidazolyl-reactive or guanidinyl-reactive).

Any convenient methods and reagent may be adapted for use in the subjectlabelling methods in order to covalently link the conjugation tag to thetarget molecule. Methods of interest for labelling a target, include butare not limited to, those methods and reagents described by Hermanson,Bioconjugate Techniques, Third edition, Academic Press, 2013. Thecontacting step may be performed in an aqueous solution. In someinstances, the conjugation tag includes an amino functional group andthe target molecule includes an activated ester functional group, suchas a NHS ester or sulfo-NHS ester, or vice versa. In certain instances,the conjugation tag includes a maleimide functional group and the targetmolecule includes a thiol functional group, or vice versa. In certaininstances, the conjugation tag includes an alkyne (e.g., a cyclooctynegroup) functional group and the target molecule includes an azidefunctional group, or vice versa, which can be conjugated via Clickchemistry.

Any convenient target molecules may be selected for labelling utilizingthe subject methods. Target molecules of interest include, but are notlimited to, a nucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA orANA molecule, a protein, such as a fusion protein, a modified protein,such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, oracetylated protein, or an antibody, a peptide, an aggregatedbiomolecule, a cell, a small molecule, a vitamin and a drug molecule. Asused herein, the term “a target protein” refers to all members of thetarget family, and fragments thereof. The target protein may be anyprotein of interest, such as a therapeutic or diagnostic target,including but not limited to: hormones, growth factors, receptors,enzymes, cytokines, osteoinductive factors, colony stimulating factorsand immunoglobulins. The term “target protein” is intended to includerecombinant and synthetic molecules, which can be prepared using anyconvenient recombinant expression methods or using any convenientsynthetic methods, or purchased commercially. In some embodiments, thetarget molecule is a specific binding member (e.g., as describedherein). In certain instances, the specific binding member is anantibody. In some instances, the specific binding member is an antibodyfragment or binding derivative thereof. In some case, the antibodyfragment or binding derivative thereof is selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody anda triabody.

In some cases, the method includes a separating step where the labelledtarget molecule is separated from the reaction mixture, e.g., excessreagents or unlabeled target. A variety of methods may be utilized toseparate a target from a sample, e.g., via immobilization on a support,precipitation, chromatography, and the like.

In some instances, the method further includes detecting and/oranalyzing the labelled target molecule. In some instances, the methodfurther includes fluorescently detecting the labelled target molecule.Any convenient methods may be utilized to detect and/or analyze thelabelled target molecule in conjunction with the subject methods andcompositions. Methods of analyzing a target of interest that find use inthe subject methods, include but are not limited to, flow cytometry,fluorescence microscopy, in-situ hybridization, enzyme-linkedimmunosorbent assays (ELISAs), western blot analysis, magnetic cellseparation assays and fluorochrome purification chromatography.Detection methods of interest include but are not limited tofluorescence spectroscopy, fluorescence microscopy, nucleic acidsequencing, fluorescence in-situ hybridization (FISH), protein massspectroscopy, flow cytometry, and the like.

Detection may be achieved directly via the polymeric dye or polymerictandem dye, or indirectly by a secondary detection system. The lattermay be based on any one or a combination of several different principlesincluding, but not limited to, antibody labelled anti-species antibodyand other forms of immunological or non-immunological bridging andsignal amplification systems (e.g., biotin-streptavidin technology,protein-A and protein-G mediated technology, or nucleic acidprobe/anti-nucleic acid probes, and the like). Suitable reportermolecules may be those known in the field of immunocytochemistry,molecular biology, light, fluorescence, and electron microscopy, cellimmunophenotyping, cell sorting, flow cytometry, cell visualization,detection, enumeration, and/or signal output quantification. More thanone antibody of specific and/or non-specific nature might be labelledand used simultaneously or sequentially to enhance target detection,identification, and/or analysis.

Methods of Preparation

Aspects of the present disclosure include methods of preparing thesubject multichromophores and polymeric tandem dyes. One advantage ofthe subject multichromophores is the modularity of the underlyingscaffold. In some instances, the modular scaffold is a linear polymerhaving a defined sequence of repeat units. The pendant chromophores usedcan be chosen from a wide range of dyes which can be covalently attachedto the polymeric backbone, either pre- or post-synthesis of the polymer.

In some embodiments, polymeric backbones of the subjectmultichromophores (see e.g., formula (IX) as described herein) can beprepared using cyclic carbonate or protected carbonate monomers. Methodsand co-monomers of interest include, but are not limited to, thosedescribed by Barnes et al. in WO2013036532, Cooley et al. (J. Am. Chem.Soc., 131, 45, 1640-3, 2009), and Rothbard et al. in U.S. Pat. No.7,169,814. Such monomers can be utilized in a polymerization reactionusing an initiator and a suitable feed ratio of cyclic carbonatemonomers to provide a polymeric backbone. Alternative, protectedcarbonate monomers can be assembled in a step wise synthesis to providea defined sequence.

Co-monomers of interest can be linked using compatible chemoselectivefunctional groups and chemistries. In some instances, co-monomers arelinked via Click chemistry, such as copper catalyzed azide-alkynecycloaddition, strain-promoted azide-alkyne cycloaddition, alkene azide[3+2] cycloaddition, alkene and tetrazine inverse demand Diers-Alder,Staudinger ligation and the like (see e.g., Kolb et al., Angew Chem IntEd Engl. 40:2004-2021, 2001). As such, linking of co-monomer can beachieved via conjugations of pairs of compatible chemoselectivefunctional groups such as, alkyne/azide, tetrazine/alkene, azide/alkene,phosphine/azide, and the like. Non-limiting examples of azide-alkynecycloaddition reactions include copper-catalyzed azide-alkynecycloaddition (CuAAC) reactions and ruthenium-catalyzed azide-alkynecycloaddition (RuAAC) reactions. CuAAC works over a broad temperaturerange, is insensitive to aqueous conditions and a pH range over 4 to 12,and tolerates a broad range of functional groups (see Himo et al, J AmChem Soc. 127:210-216, 2005). The active Cu(I) catalyst can begenerated, for example, from Cu(I) salts or Cu(II) salts using sodiumascorbate as the reducing agent. This reaction forms 1,4-substitutedproducts. RuAAC utilizes pentamethylcyclopentadienyl ruthenium chloride[Cp*RuCl] complexes that are able to catalyze the cycloaddition ofazides to terminal alkynes, regioselectively leading to1,5-disubstituted 1,2,3-triazoles (see Rasmussen et al., Org. Lett.9:5337-5339, 2007). Further, and in contrast to CuAAC, RuAAC can also beused with internal alkynes to provide fully substituted 1,2,3-triazoles.

In some embodiments, polymeric backbones of the subjectmultichromophores are polypeptides. Any convenient peptide synthesismethods can be utilized in the preparation of such polymeric backbonesof the subject multichromophores. In some cases, solid phase peptidesynthesis (SPPS) methods are utilized to prepare polymeric backbones ofthe subject multichromophores via a defined stepwise synthesis.Conventional protecting group strategies provide for deprotection andcoupling of amino acid residues of interest in a defined sequence, whilesidechain functional groups of interest can be orthogonally protected.In some cases, a Fmoc/tert-butyl methodology is utilized. In some cases,a Boc/benzyl methodology is utilized. Such protecting group strategiescan also be adapted for use in the installation of pendant groups ofinterest on the sidechains of particular amino acid residues, and/or atthe polypeptide terminals. In certain instances, one or more of thependant groups can be installed into the protected amino acid monomerstarting materials. In some cases, the pendant groups are installed intothe multichromophore after SPPS of the polymeric backbone has beenperformed. By use of orthogonal protecting groups, different groups ofinterest can be independently installed onto the polypeptide backbone,e.g., at the N- and C-terminals, and/or at each distinct type of aminoacid residue. The polypeptide can include β2-amino acid residues,β3-amino acid residues, α-amino acid residues, or mixtures thereof.

In some embodiments, the method of preparing a light harvestingmultichromophore (e.g., as described herein) includes synthesizing aprotected polypeptide having a defined amino acid sequence consisting ofblocks of first amino acid residues separated by single occurrences ofsecond amino acid residues. As described herein, the preparation of adefined sequence of amino acid residues can provide for a desiredconfiguration of pendant donor chromophores and acceptor fluorophoresalong the polypeptide. When installation of the pendant groups isachieved post polypeptide synthesis, chemoselective functional groups ofthe amino acid sidechains along the polypeptide backbone are selectivelyconjugated to pendant groups. In certain instances, the light harvestingmultichromophore is a polypeptide having a defined sequence wherein:each block of first amino acid residues comprises at least two residues;the first amino acid residues each comprise a protected firstchemoselective sidechain group; and the second amino acid residues eachcomprise a protected second chemoselective sidechain group.

Alternatively, installation of the pendant donor chromophore groups canbe achieved by utilizing protected amino acid building blocks thatalready include the chromophore group as part of the sidechain. It isunderstood that the pendant chromophore groups can be installed first onat least the sidechains of a defined polypeptide sequence to produce alight harvesting multichromophore. A variety of additional pendantgroups including acceptor fluorophores can then be installedsequentially via selective conjugation(s) to chemoselective tag(s)attached to particular residues of the sequence.

In some cases, the method further includes coupling reactive acceptorfluorophore moieties to deprotected second chemoselective sidechaingroups of the second amino acid residues to produce pendant acceptorfluorophores.

In certain instances of the method, the defined amino acid sequencecomprises one or more amino acid sequence segments selected from thefollowing:

XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXXXXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X)_(n)YXY(X)_(n)YX XXY(X)_(n)YXX XXXY(X)_(n)YXXX XXXXY(X)_(n)YXXXXXXXXXY(X)_(n)YXXXXXwherein each X is a first amino acid residue having a firstchemoselective functional group, or protected version thereof, and eachY is a second amino acid residue having a second chemoselectivefunctional group, or protected version thereof.

In certain embodiments of the sequences above, each X is a lysine orornithine residue, or a protected version thereof, having a sidechainamino group that can be selectively covalently N-linked to a pendantdonor chromophore group; and each Y is a cysteine residue or a protectedcysteine residue having a sidechain thiol group that can be selectivelycovalently linked to a pendant acceptor fluorophore.

Polypeptide sequences comprising two or more of the sequence segmentsdescribed above can be prepared, optionally separated by additionalthird amino acid residues (e.g., (segment 1)-Z-(segment 2) where Z isthe third residue). This third amino acid residue can be a spacerresidue (e.g., not having a sidechain or a tag), or a residue having achemoselective functional group suitable for selective installation ofan additional moiety of interest, such as a second pendant lightabsorbing chromophore, a chemoselective tag (e.g., a bio-orthogonalclick chemistry tag), a linker, a linked biomolecule, a acceptorfluorophore, a WSG, etc. In certain instances the additional moiety ofinterest is incorporated into the third amino acid residue prior topolypeptide synthesis.

Similarly, a variety of moieties can be installed at the N- and/orC-terminal of the polypeptide during or after peptide synthesis. Incertain instances, the method further includes deprotecting theN-terminal of the protected polypeptide and coupling a G1 group (e.g.,as described herein) to the N-terminal of the N-terminal deprotectedpolypeptide. G1 can be any convenient terminal group (e.g., a cappinggroup such as an alkanoyl, e.g., acetyl), a donor chromophore group, alinker having a particular chemoselective tag or a biomolecule ofinterest.

In some cases, the method further includes installing a G2 group at theC-terminal of the polypeptide. This can be achieved in a variety ofways: e.g., during SPPS where the C-terminal group G2 is installedbetween the solid support and the first amino acid residue of thesequence; during cleavage of the polypeptide from the solid support; orpost synthesis where a moiety of interest (e.g., a specific bindingmember) or a particular chemoselective tag directed to same, can becoupled to the C-terminal of the polypeptide. In some instances, nativechemical ligation methods can be utilized to prepare a C-terminalthioester polypeptide suitable for coupling to a polypeptide fragment ora moiety of interest.

Any of the polypeptide multichromophores described herein can beprepared according to the subject methods, e.g., polypeptides offormulae (IV)-(VII), and synthetic precursors thereof, as describedherein.

A summary of some of the various methods available for synthesizing thesubject polypeptide multichromphores can be found in Steward et al., in“Solid Phase Peptide Synthesis”, W.H. Freeman Co., San Francisco, 1969;Bodanszky et al., in “Peptide Synthesis”, John Wiley & Sons, SecondEdition, 1976 and Meienhofer, in “Hormonal Proteins and Peptides”, Vol.2, p. 46, Academic Press (New York), 1983; and Kent, Ann. Rev. Biochem.,57, 957, 1988, for solid phase peptide synthesis, and Schroder et al.,in “The Peptides”, Vol. 1, Academic Press (New York), 1965 for solutionsynthesis. Any convenient protecting group strategies may be used suchas, but are not limited to, Fmoc solid-phase peptide synthesis and Bocsolid-phase peptide synthesis strategies. In Boc solid-phase peptidesynthesis a Boc-amino protecting group is used at the amino terminal andbenzyl or benzyl-based or other convenient protecting groups may be usedfor the protection of sidechain functional groups. In Fmoc solid-phasepeptide synthesis a Fmoc-amino protecting group is used at the aminoterminal and tert-butyl or benzyl-based or other convenient protectinggroups may be used for protection of sidechain functional groups.Convenient protecting groups that may be used in such synthetic methodsare described in the above references and by McOmic in “ProtectiveGroups in Organic Chemistry”, Plenum Press, New York, 1973; and Greeneand Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons,4th Edition, 2006.

Systems

Aspects of the invention further include systems for use in practicingthe subject methods and compositions. A sample analysis system caninclude sample field of view or a flow channel loaded with a sample anda labelled specific binding member. In some embodiments, the system is aflow cytometric system including: a flow cytometer including a flowpath; a composition in the flow path, wherein the composition includes:a sample; and a labelled specific binding member (e.g., as describedherein). In some embodiments, the system for analyzing a sample is afluorescence microscopy system, including: a fluorescence microscopecomprising a sample field of view; and a composition disposed in thesample field of view, wherein the composition comprises a sample; and alabelled specific binding member (e.g., as described herein).

In some instances of the systems, the labelled specific binding memberincludes: a water solvated light harvesting multichromophore (e.g., asdescribed herein) and a specific binding member that specifically bindsa target analyte covalently linked to the multichromophore. In somecases, the labelled specific binding member further comprises asignaling chromophore covalently linked to the multichromophore of thepolymeric dye in energy-receiving proximity therewith. In some instancesof the subject systems, the labelled specific binding member, themultichromophore is described by any one of formulae (I)-(IX) (e.g., asdescribed herein), wherein: G¹ and G² are each independently selectedfrom the group consisting of a terminal group, a polymeric conjugatedsegment, a linker and a linked specific binding member, wherein at leastone of G¹ and G² is a linked specific binding member.

In certain embodiments of the systems, the composition further includesa second specific binding member that is support bound and specificallybinds the target analyte. In some cases, the support includes a magneticparticle. As such, in certain instances, the system may also include acontrollable external paramagnetic field configured for application toan assay region of the flow channel.

The sample may include a cell. In some instances, the sample is acell-containing biological sample. In some instances, the sampleincludes a labelled specific binding member specifically bound to atarget cell. In certain instances, the target analyte that isspecifically bound by the specific binding member is a cell surfacemarker of the cell. In certain cases, the cell surface marker isselected from the group consisting of a cell receptor and a cell surfaceantigen.

In certain aspects, the system may also include a light sourceconfigured to direct light to an assay region of the flow channel orsample field of view. The system may include a detector configured toreceive a signal from an assay region of the flow channel or a samplefield of view, wherein the signal is provided by the fluorescentcomposition. Optionally further, the sample analysis system may includeone or more additional detectors and/or light sources for the detectionof one or more additional signals.

In certain aspects, the system may further include computer-basedsystems configured to detect the presence of the fluorescent signal. A“computer-based system” refers to the hardware means, software means,and data storage means used to analyze the information of the presentinvention. The minimum hardware of the computer-based systems of thepresent invention includes a central processing unit (CPU), input means,output means, and data storage means. A skilled artisan can readilyappreciate that any one of the currently available computer-based systemare suitable for use in the subject systems. The data storage means mayinclude any manufacture including a recording of the present informationas described above, or a memory access means that can access such amanufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g., word processing text file, database format, etc.

A “processor” references any hardware and/or software combination thatwill perform the functions required of it. For example, any processorherein may be a programmable digital microprocessor such as available inthe form of an electronic controller, mainframe, server or personalcomputer (desktop or portable). Where the processor is programmable,suitable programming can be communicated from a remote location to theprocessor, or previously saved in a computer program product (such as aportable or fixed computer readable storage medium, whether magnetic,optical or solid state device based). For example, a magnetic medium oroptical disk may carry the programming, and can be read by a suitablereader communicating with each processor at its corresponding station.

In addition to the sensor device and signal processing module, e.g., asdescribed above, systems of the invention may include a number ofadditional components, such as data output devices, e.g., monitorsand/or speakers, data input devices, e.g., interface ports, keyboards,etc., fluid handling components, power sources, etc.

In certain aspects, the system includes a flow cytometer. Suitable flowcytometer systems and methods for analyzing samples include, but are notlimited to those described in Ormerod (ed.), Flow Cytometry: A PracticalApproach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), FlowCytometry Protocols, Methods in Molecular Biology No. 91, Humana Press(1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, etal. (2012) Ann Clin Biochem. January; 49(pt 1):17-28; Linden, et. al.,Semin Throm Hemost. 2004 October; 30(5):502-11; Alison, et al. J Pathol,2010 December; 222(4):335-344; and Herbig, et al. (2007) Crit Rev TherDrug Carrier Syst. 24(3):203-255; the disclosures of which areincorporated herein by reference. In certain instances, flow cytometrysystems of interest include BD Biosciences FACSCanto™ flow cytometer, BDBiosciences FACSVantage™, BD Biosciences FACSort™, BD BiosciencesFACSCount™, BD Biosciences FACScan™, and BD Biosciences FACSCalibur™systems, BD Biosciences Accuri™ systems, BD Biosciences FACSCanto™systems, BD Biosciences FACSCelesta™ systems, BD Biosciences FACSLyric™systems, BD Biosciences FACSVerse™ systems, BD Biosciences FACSymphony™systems, BD Biosciences LSRFortessa™ systems, BD Biosciences Influx™cell sorter, BD Biosciences FACSJazz™ cell sorter and BD BiosciencesFACSAria™ cell sorter, BD Biosciences FACSMelody™ cell sorter, and thelike.

In certain embodiments, the subject systems are flow cytometer systemswhich incorporate one or more components of the flow cytometersdescribed in U.S. Pat. Nos. 3,960,449; 4,347,935; 4,667,830; 4,704,891;4,770,992; 5,030,002; 5,040,890; 5,047,321; 5,245,318; 5,317,162;5,464,581; 5,483,469; 5,602,039; 5,620,842; 5,627,040; 5,643,796;5,700,692; 6,372,506; 6,809,804; 6,813,017; 6,821,740; 7,129,505;7,201,875; 7,544,326; 8,140,300; 8,233,146; 8,753,573; 8,975,595;9,092,034; 9,095,494 and 9,097,640; the disclosures of which are hereinincorporated by reference.

Other systems may find use in practicing the subject methods. In certainaspects, the system may be a fluorimeter or microscope loaded with asample having a fluorescent composition of any of the embodimentsdiscussed herein. The fluorimeter or microscope may include a lightsource configured to direct light to the assay region of the flowchannel or sample field of view. The fluorimeter or microscope may alsoinclude a detector configured to receive a signal from an assay regionof the flow channel or field of view, wherein the signal is provided bythe fluorescent composition.

Kits

Aspects of the invention further include kits for use in practicing thesubject methods and compositions. The compositions of the invention canbe included as reagents in kits either as starting materials or providedfor use in, for example, the methodologies described above.

A kit can include a polymeric dye including a water solvated lightharvesting multichromophore (e.g., as described herein) and a container.Any convenient containers can be utilized, such as tubes, bottles, orwells in a multi-well strip or plate, a box, a bag, an insulatedcontainer, and the like. The subject kits can further include one ormore components selected from a polymeric tandem dye, a fluorophore, aspecific binding member, a specific binding member conjugate, a supportbound specific binding member, a cell, a support, a biocompatibleaqueous elution buffer, and instructions for use. In some embodiments ofthe kit, the multichromophore is covalently linked to a specific bindingmember. In some instances, the specific binding member is an antibody.In certain instances, the specific binding member is an antibodyfragment or binding derivative thereof. In certain cases, the antibodyfragment or binding derivative thereof is selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody anda triabody.

In certain embodiments, the kit finds use in evaluating a sample for thepresence of a target analyte, such as an intracellular target. As such,in some instances, the kit includes one or more components suitable forlysing cells. The one or more additional components of the kit may beprovided in separate containers (e.g., separate tubes, bottles, or wellsin a multi-well strip or plate).

In certain aspects, the kit further includes reagents for performing aflow cytometric assay. Reagents of interest include, but are not limitedto, buffers for reconstitution and dilution, buffers for contacting acell sample the multichromophore, wash buffers, control cells, controlbeads, fluorescent beads for flow cytometer calibration and combinationsthereof. The kit may also include one or more cell fixing reagents suchas paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or anycombinations or buffers thereof. Further, the kit may include a cellpermeabilizing reagent, such as methanol, acetone or a detergent (e.g.,triton, NP-40, saponin, tween 20, digitonin, leucoperm, or anycombinations or buffers thereof. Other protein transport inhibitors,cell fixing reagents and cell permeabilizing reagents familiar to theskilled artisan are within the scope of the subject kits.

The compositions of the kit may be provided in a liquid composition,such as any suitable buffer. Alternatively, the compositions of the kitmay be provided in a dry composition (e.g., may be lyophilized), and thekit may optionally include one or more buffers for reconstituting thedry composition. In certain aspects, the kit may include aliquots of thecompositions provided in separate containers (e.g., separate tubes,bottles, or wells in a multi-well strip or plate).

In addition, one or more components may be combined into a singlecontainer, e.g., a glass or plastic vial, tube or bottle. In certaininstances, the kit may further include a container (e.g., such as a box,a bag, an insulated container, a bottle, tube, etc.) in which all of thecomponents (and their separate containers) are present. The kit mayfurther include packaging that is separate from or attached to the kitcontainer and upon which is printed information about the kit, thecomponents of the and/or instructions for use of the kit.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, DVD, portable flash drive, etc., on which theinformation has been recorded. Yet another means that may be present isa website address which may be used via the Internet to access theinformation at a removed site. Any convenient means may be present inthe kits.

Utility

The polymeric dyes, compositions, methods and systems as describedherein may find use in a variety of applications, including diagnosticand research applications, in which the labelling, detection and/oranalysis of a target of interest is desirable. Such applications includemethodologies such as cytometry, microscopy, immunoassays (e.g.competitive or non-competitive), assessment of a free analyte,assessment of receptor bound ligand, and so forth. The compositions,system and methods described herein may be useful in analysis of any ofa number of samples, including but not limited to, biological fluids,cell culture samples, and tissue samples. In certain aspects, thecompositions, system and methods described herein may find use inmethods where analytes are detected in a sample, if present, usingfluorescent labels, such as in fluorescent activated cell sorting oranalysis, immunoassays, immunostaining, and the like. In certaininstances, the compositions and methods find use in applications wherethe evaluation of a sample for the presence of a target analyte is ofinterest.

In some cases, the methods and compositions find use in any assay formatwhere the detection and/or analysis of a target from a sample is ofinterest, including but not limited to, flow cytometry, fluorescencemicroscopy, in-situ hybridization, enzyme-linked immunosorbent assays(ELISAs), western blot analysis, magnetic cell separation assays andfluorochrome purification chromatography. In certain instances, themethods and compositions find use in any application where thefluorescent labelling of a target molecule is of interest. The subjectcompositions may be adapted for use in any convenient applications wherepairs of specific binding members find use, such as biotin-streptavidinand hapten-anti-hapten antibody.

EXAMPLES Example 1

The synthesis of the subject polymeric dyes can be achieved by a Suzukicoupling method. Other methods such as a C—H bond arylation or a Stillecoupling method can also be utilized to construct and polymerize arepeat unit including an aryl or heteroaryl co-monomer with a saturatedPEG co-monomer.

The formula of a tandem dye based on multichromophore A (MC) is depictedin FIG. 1. A series of tandem dyes having the following core structurewith different acceptor fluorophores (“Dye”) was prepared:

The spectroscopic properties of the exemplary polymeric tandem dyes wasevaluated, as shown in FIG. 2 and FIG. 3.

TABLE 1 Spectroscopic data for a pendant multichromophore andcorresponding tandem dyes λmax_((em)) Quantum Amplification Emitter (nm)D/A Yield (%) factor MC 523 N/A  9 N/A MC-dye 1 649 0.09 12 5.1 MC-dye 2661 0.06 23 2.1 MC-dye 3 682 0.06 33 1.8 MC-dye 4 717 0.08 27 2.2 MC-dye5 740 0.12 16 2.3 MC-dye 6 786 0.16 14 2.1

Example 2

Solid phase peptide synthesis methods were used to prepare a sequence oflysine and cysteine amino acid residues KCKK, as shown in FIG. 4.Carboxylic acid-substituted BODIPY groups were conjugated to theN-terminal and to the sidechain amino groups of the lysine residues ofthe peptide via amide bond coupling. A maleimide-substituted acceptorfluorophore (Dye 7) was conjugated to the cysteine residue utilizingmaleimide-thiol coupling chemistry. The peptide was prepared with aC-terminal linker suitable for conjugation to a biomolecule, e.g.,protein.

The spectroscopic properties of the BODIPY-based tandem dye of FIG. 4were characterized. FIG. 5A shows absorption and emission spectra forthe BODIPY donor pendant group. FIG. 5B shows absorption and emissionspectra for the acceptor dye (Dye 7). FIG. 5C shows absorption andemission spectra for the exemplary polymeric tandem dye of FIG. 4. Forthe emission spectrum of the scaffolded chromophore, the primarychromophore is being excited. For this system, the emission of the donoris nearly completely quenched and most of the emission is that of theacceptor fluorophore. The quantum yield of the polymeric tandem dye iscomparable to the acceptor fluorophore (Dye 7), suggesting the tandemdye exhibits an efficient energy transfer process. The fluorescence ofthis exemplary tandem dye was amplified by approximately 2-fold overthat of the acceptor fluorophore alone (Dye 7).

Polypeptides providing a ratio of donor to acceptor of 5:1. 6:1, 7:1,8:1, 9:1, 10:1 and 20:2 are prepared and characterized using the methodsdescribed above.

Example 3

Water-soluble fluorescent polymer dyes can be prepared by clickpolymerization under mild conditions. These polymers dyes can becovalently bonded to a bio-recognition unit (biomolecule) and serve asfluorescent probes for a target. When secondary dyes are incorporated asacceptors, polymeric tandem dyes are formed. By tuning color of theacceptor dyes, polymeric tandem dyes having the same excitationwavelength (see FIG. 9A) but a range of different alternative emissionwavelengths can be prepared (see FIG. 9B).

PEGylated BODIPY dyes with desired narrow absorption are selected asfluorophore pendant dyes. The pendant fluorophore is chemically boundedto water soluble ethylene glycol ether monomers terminated with diazide,dialkyne, or azide/alkyne. The PEG polymer chain with attached BODIPYdyes can be prepared using Cu(I) catalyzed 1,3-dipolar cycloadditions ofalkyne to azide in good MW (e.g., as described herein). When only onetype of fluorophore dye is attached as a pendant group, the resultedpolymer exhibits fluorescence as a simple fluorescent polymer dye (seeFIG. 8). When a second acceptor fluorophore is attached, a polymer chainwith two fluorophore pedants is achieved. One fluorophore at higherenergy level can serve as an energy donor and the 2nd fluorophore at thelow energy level as energy acceptor. By mixing the suitable pair andratio of donor/acceptor, the polymeric tandem dyes with desired emissionwavelength can be obtained.

The following polymeric dyes are prepared by click polymerizationmethods, using co-monomers and methods as described by the syntheticschemes of FIGS. 7A and 7B, where it is understood that n and m togethercan represent the average length of the polymer and the relative ratiosof co-monomers, and that n and m can be selected as desired bycontrolling the parameters of the polymerization reaction using anyconvenient methods. It is understood that the depending on the method ofpolymerization, the n and m repeating units may be present in a randomconfiguration or in a block configuration. In addition, the followingpolymeric structures can be further conjugated to any convenientmolecule of interest, e.g., a biomolecule, using Click chemistry via aterminal azide or alkyne group. In some cases, n and m are eachindependently selected from 1 to 1000, such as 1 to 500, 1 to 200, 1 to100, 2 to 100 or 5 to 100.

FIG. 8 shows the absorption and emission spectra of a base polymer dyeincluding a pendant BODIPY dye of the general structure shown above.Other exemplary polymeric dyes where R is a linked donor dye (e.g., aBODIPY dye as described above), and including a sidechain —NH₂ groupsuitable for conjugation (e.g., via an amide bond and linker to anacceptor dye) are:

Exemplary polymeric tandem dye structures are shown below where R is alinked donor dye (e.g., a BODIPY dye as described above), and includinga linked acceptor dye of interest (e.g., suitable for conjugation (e.g.,to an acceptor dye) such as DY-754 or DY-704, where DY refers to aDyomics dyes having, e.g., emission maximum wavelengths of 754 nm or 704nm, respectively. By selecting an acceptor dye of interest that overlapsat least partially with the emission spectrum of the base polymer (seeFIG. 8), polymeric tandem dyes having a variety of emission wavelengthscan be produced. See e.g., FIG. 9A-9B.

Additional Embodiments

Notwithstanding the appended claims, the disclosure set forth herein isalso defined by the following clauses:

Clause 1. A water-soluble light harvesting multichromophore comprising:

a polymeric backbone comprising non-conjugated repeat units; and

a plurality of pendant donor chromophore groups each independentlylinked to a non-conjugated repeat unit of the polymeric backbone.

Clause 2. The multichromophore according to clause 1, wherein thependant donor chromophore groups are each substituted with a watersoluble group.

Clause 3. The multichromophore according to clause 1, wherein thepolymeric backbone is a linear polymer.

Clause 4. The multichromophore according to clause 1, wherein thependant donor chromophore groups are configured in energy-transferringproximity to each other.

Clause 5. The multichromophore according to clause 4, wherein theconfiguration of pendant donor chromophore groups exhibits, uponexcitation with incident light, quenching of fluorescence relative to anunquenched isolated donor chromophore group.

Clause 6. The multichromophore according to any one of clauses 1-5,wherein the pendant donor chromophore groups are selected from a fusedtricyclic aryl or heteroaryl group and a BODIPY group.

Clause 7. The multichromophore according to clause 6, wherein thependant donor chromophore groups are selected from fluorene, carbazoleand silole.

Clause 8. The multichromophore according to any one of clauses 6-7,wherein the pendant donor chromophore groups are a fused tricyclic arylor heteroaryl group having one of the following formulae:

wherein:

* is a point of linkage to a non-conjugated repeat unit of the polymericbackbone;

Y is C(R¹³)₂, —C(R¹³)₂C(R¹³)₂—, —C(R¹³)₂Si(R¹³)₂—, NR¹³, Si(R¹³)₂ or Se;

each Z is independently CH, CR or N;

each R¹³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, a WSG and -L¹¹-Z¹,wherein L¹¹ is a linker and Z¹ is a non-conjugated repeat unit, orwherein any two convenient R′ groups are optionally cyclically linked;and

each R is independently H or one or more substituents and wherein anytwo convenient R groups are optionally cyclically linked;

wherein one of R and R¹³ is linked to a non-conjugated repeat unit ofthe polymeric backbone.

Clause 9. The multichromophore according to any one of clauses 1-6,wherein the pendant donor chromophore groups are BODIPY groups.

Clause 10. The multichromophore according to clause 9, wherein theBODIPY groups are described by the formula:

wherein:

R¹-R⁷ are each independently selected from H, alkyl, substituted alkyl,alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, water solubilizing group (WSG) and -L¹-Z¹, or

-   -   optionally any one or more pairs of substituents selected from        R⁶ and R⁷, R² and R³, R⁵ and R⁶, R³ and R⁴, R⁴ and R¹ and R⁵ and        R¹, together form a divalent radical and are cyclically linked        and together with the carbon atoms to which they are bound        provide a 5- or 6-membered fused heterocycle, carbocycle, aryl        or heteroaryl ring (e.g., a 5- or 6-membered ring comprising        carbon atoms and 0-3 heteroatoms selected from O, S and N),        which ring may be unsubstituted or further substituted with a        substituent independently selected from alkyl, substituted        alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, alkenyl, substituted        alkenyl, alkynyl, substituted alkynyl, water solubilizing group        (WSG) and -L¹-Z¹;

L¹ is a linker;

Z¹ is a non-conjugated repeat unit of the polymeric backbone; and

Y¹ and Y² are independently selected from F, OH, H, cyano, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl and WSG;

wherein one of Y¹, Y² and R¹-R⁷ is linked to a non-conjugated repeatunit of the polymeric backbone.

Clause 11. The multichromophore according to clause 10, wherein R¹ is anoptionally substituted aryl or heteroaryl linked to a non-conjugatedrepeat unit of the polymeric backbone.

Clause 12. The multichromophore according to any one of clauses 10-11,wherein Y¹ and Y² each comprise a water solubilizing group (WSG).

Clause 13. The multichromophore according to any one of clauses 10-12,wherein the pendant donor chromophore groups are described by thefollowing structure:

wherein:

* is a point of linkage to a non-conjugated repeat unit of the polymericbackbone;

Y¹ and Y² are each alkynyl substituted with a WSG.

Clause 14. The multichromophore according to clause 13, wherein thependant donor chromophore groups are described by the followingstructure:

wherein:

R¹¹ is L¹-Z¹ (a linked non-conjugated repeat unit of the polymericbackbone);

each R⁹ is an optional substituent selected from halogen, hydroxyl,cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy,aryl, substituted aryl, heteroaryl and substituted heteroaryl; and t is0-4.

Clause 15. The multichromophore according to any one of clauses 1-14,wherein the pendant donor chromophore groups are substituted with one ormore water solubilizing groups (WSGs) independently selected from thefollowing formula:

wherein:

T⁵ is an optional linker;

each T⁶ is an linker;

R¹¹ and R are independently H, alkyl or substituted alkyl; and

each s is an integer from 1 to 50.

Clause 16. The multichromophore according to any one of clauses 1-15,comprising a segment of the formula:

wherein:

each M¹ and M² is independently an aryl or heteroaryl co-monomer;

each S¹ and S² is independently a non-conjugated spacer unit;

each D¹ is independently a pendant donor chromophore linked to M¹;

each Z¹ is independently a chemoselective tag linked to M²;

x is 75 mol % or more; and

y is 25 mol % or less.

Clause 17. The multichromophore according to clause 16, wherein theM¹-S¹ and M²-S² repeat units of the polymeric backbone have a randomconfiguration.

Clause 18. The multichromophore according to any one of clauses 16-17,wherein:

each M¹ and M² independently comprises one or more groups selected fromfluorene, carbazole, silole, biphenylene and phenylene; and

each S¹ and S² is independently a saturated spacer unit selected from adivalent polyethylene glycol (PEG) and a divalent modified PEG group.

Clause 19. The multichromophore according to any one of clauses 1-15,comprising a segment of the formula:

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹ andSM² co-monomers that are each independently a saturated non-conjugatedco-monomer;

each D¹ is independently a pendant donor chromophore linked to SM¹;

each Z¹ is independently a chemoselective tag linked to SM²;

x is 75 mol % or more; and

y is 25 mol % or less.

Clause 20. The multichromophore according to clause 19, wherein the SM¹and SM² repeat units of the polymeric backbone have a randomconfiguration.

Clause 21. The multichromophore according to any one of clauses 19-20,wherein SM¹ and SM² are co-monomers derived from an acrylate, amethacrylate, an acrylamide, a polystyrene, a ROMP monomer, an ADMETmonomer or a cyclic carbonate.

Clause 22. The multichromophore according to clause 21, wherein SM¹ andSM² are selected from the following formulae:

wherein:

R²¹ is -L¹-D¹ or -L²-Z¹;

D¹ is the pendant donor chromophore linked to M¹;

Z¹ is a chemoselective tag linked to M²;

L¹ and L² are optional linkers;

X is O or NR″;

R²² is H or lower alkyl;

R″ is H or lower alkyl, substituted lower alkyl and WSG; and

* is a connection to the polymeric backbone.

Clause 23. The multichromophore according to clause 19, wherein themultichromophore is of formula (XXI):

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹, SM²and SM³ co-monomers that are each linked via a group T that is theproduct of a click chemistry or chemoselective group conjugationreaction;

SM³ optionally comprises a linked WSG;

each D¹ is independently a pendant light absorbing chromophore linked toSM¹;

each Z¹ is independently a chemoselective tag linked to SM²;

x is 50 mol % or more; and

y+z is 50 mol % or less, where * is a connection to the polymericbackbone of the multichromophore or a terminal group.

Clause 24. The multichromophore according to clause 23, wherein SM¹, SM²and SM³ comprise the following structures:

wherein:

each X is independently 0 or NR³¹ wherein R³¹ is H, alkyl, substitutedalkyl, alkanoyl or substituted alkanoyl;

each r and s is independently 1-6 (e.g., 1, 2 or 3);

each d and e is independently 1-12 (e.g., 1-6, such as 1, 2, 3, 4, 5 or6);

t is 0 or 1;

D¹ is a pendant donor chromophore;

Z¹ is a chemoselective tag (e.g., as described herein);

WSG is a water solubilizing group (e.g., as described herein);

each L¹, L² and L³ is independently a linker; and

* is a connection to a 1,4-substituted 1,2,3-triazole (T) having one ofthe following structures:

or a terminal group G¹ or G² (e.g., as described herein).

Clause 25. The multichromophore according to clause 19, wherein therepeat units of the polymeric backbone have a defined linear sequence.

Clause 26. The multichromophore according to clause 25, wherein SM¹ andSM² are co-monomers derived from amino acids, peptoid monomers, aprotected carbonate monomer or a cyclic carbonate monomer.

Clause 27. The multichromophore according to any one of clauses 19, 25and 26, wherein the polymeric backbone is a polypeptide having a definedsequence of α-amino acid residues and/or β-amino acid residues.

Clause 28. The multichromophore according to any one of clauses 19 and25-27, wherein the multichromophore is of the formula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each Z¹ is independently a chemoselective tag;

each L¹ and L² are independently a linker;

p₁ and q₁ are independently 0 or 1 wherein p₁+q₁≤1;

p₂ and q₂ are independently 0 or 1 wherein p₁+q₁≤1;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a donor chromophore group, an acceptor fluorophoregroup, a linker and a linked specific binding member.

Clause 29. The multichromophore of clause 28, wherein p₁ and p₂ are each0 and q₁ and q₂ are each 1.

Clause 30. The multichromophore of clause 28, wherein p₁ and p₂ are each1 and q₁ and q₂ are each 0.

Clause 31. The multichromophore of clause 28, wherein p₁, p₂, q₁ and q₂are each 0 and the multichromophore is of the formula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each Z¹ is independently a chemoselective tag;

L¹ and L² are each independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a donor chromophore group, an acceptor fluorophoregroup, a linker and a linked specific binding member.

Clause 32. The multichromophore according to any one of clauses 27, 28and 31, comprising a segment of the formula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each Z¹ is independently a chemoselective tag;

each L¹ and L² are independently a linker;

n and p are each independently an integer from 1 to 20 wherein n+p 2;and

m is 1 or 2.

Clause 33. The multichromophore according to clause 32, wherein themultichromophore comprises q segments of copolymer and is of theformula:

wherein:

each (n)_(q) and each (p)_(q) is independently an integer from 1 to 20,wherein for each of the q segments (n)_(q)+(p)_(q)≥3; and

q is an integer from 1 to 100.

Clause 34. The multichromophore according to any one of clauses 19,25-28 and 31-33, wherein the polymeric backbone comprises one or moreamino acid sequences selected from the following:

XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXXXXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X)_(n)YXY(X)_(n)YX XXY(X)_(n)YXX XXXY(X)_(n)YXXX XXXXY(X)_(n)YXXXXXXXXXY(X)_(n)YXXXXXwherein:

each X is a lysine or ornithine residue covalently N-linked to a pendantdonor chromophore group; and

each Y is a cysteine residue or a protected cysteine residue.

Clause 35. The multichromophore according to any one of clauses 19, 25and 26, wherein the multichromophore has the formula:

wherein

each D¹ is independently a pendant donor chromophore group;

each Z¹ is independently a chemoselective tag;

each L¹ and L² is independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a donor chromophore group, an acceptor fluorophore, alinker and a linked specific binding member.

Clause 36. The multichromophore according to any one of clauses 19-21and 26, wherein the multichromophore has the formula:

wherein:

each D¹ is independently a pendant donor chromophore;

each Z¹ is independently a chemoselective tag;

each L¹ and L² is independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from the group consisting of aterminal group, a polymer segment, a donor chromophore group, anacceptor fluorophore, a linker and a linked specific binding member.

Clause 37. The multichromophore according to any one of clauses 16-36,wherein the pendant donor chromophore groups are BODIPY groups.

Clause 38. A polymeric tandem dye comprising:

a light harvesting multichromophore comprising:

-   -   a polymeric backbone comprising non-conjugated repeat units; and    -   a plurality of pendant donor chromophore groups each        independently linked to a non-conjugated repeat unit of the        polymeric backbone; and

an acceptor fluorophore linked to a non-conjugated repeat unit of thepolymeric backbone and configured in energy-receiving proximity to atleast one pendant donor chromophore group of the light harvestingmultichromophore.

Clause 39. The polymeric tandem dye according to clause 38, wherein thependant donor chromophore groups are each substituted with a watersoluble group.

Clause 40. The polymeric tandem dye according to clause 38, wherein thepolymeric backbone is a linear polymer.

Clause 41. The polymeric tandem dye according to clause 38, wherein thependant donor chromophore groups are configured in energy-transferringproximity to each other.

Clause 42. The polymeric tandem dye according to clause 36, having aStokes shift of 100 nm or more.

Clause 43. The polymeric tandem dye according to any one of clauses36-40, wherein the pendant donor chromophore groups are selected from afused tricyclic aryl group, a fused tricyclic heteroaryl group, and aBODIPY group.

Clause 44. The polymeric tandem dye according to clause 43, wherein thependant donor chromophore groups are selected from optionallysubstituted fluorene, carbazole and silole groups.

Clause 45. The polymeric tandem dye according to any one of clauses43-44, wherein the pendant donor chromophore groups are fused tricyclicaryl or heteroaryl groups having one of the following formulae:

wherein:

* is a point of linkage to a non-conjugated repeat unit of the polymericbackbone;

Y is C(R¹³)₂, —C(R¹³)₂C(R¹³)₂—, —C(R¹³)₂Si(R¹³)₂—, NR¹³, Si(R¹³)₂ or Se;

each Z is independently CH, CR or N;

each R¹³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, a WSG and -L¹¹-Z¹,wherein L¹¹ is a linker and Z¹ is a non-conjugated repeat unit, orwherein any two convenient R³ groups are optionally cyclically linked;and

each R is independently H or one or more substituents and wherein anytwo convenient R groups are optionally cyclically linked;

wherein one of R and R¹³ is linked to a non-conjugated repeat unit ofthe polymeric backbone.

Clause 46. The polymeric tandem dye according to clause 43, wherein thependant donor chromophore groups are BODIPY groups.

Clause 47. The polymeric tandem dye according to clause 46, wherein theBODIPY groups are described by the formula:

wherein:

R¹-R⁷ are each independently selected from H, alkyl, substituted alkyl,alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, water solubilizing group (WSG) and -L¹-Z¹, or

-   -   optionally any one or more pairs of substituents selected from        R⁶ and R⁷, R² and R³, R⁵ and R⁶, R³ and R⁴, R⁴ and R¹ and R⁵ and        R¹, together form a divalent radical and are cyclically linked        and together with the carbon atoms to which they are bound        provide a 5- or 6-membered fused heterocycle, carbocycle, aryl        or heteroaryl ring (e.g., a 5- or 6-membered ring comprising        carbon atoms and 0-3 heteroatoms selected from O, S and N),        which ring may be unsubstituted or further substituted with a        substituent independently selected from alkyl, substituted        alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, alkenyl, substituted        alkenyl, alkynyl, substituted alkynyl, water solubilizing group        (WSG) and -L¹-Z¹;

L¹ is a linker;

Z¹ is a non-conjugated repeat unit of the polymeric backbone; and

Y¹ and Y² are independently selected from F, OH, H, cyano, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl and WSG;

wherein one of Y¹, Y² and R¹-R⁷ is linked to a non-conjugated repeatunit of the polymeric backbone.

Clause 48. The polymeric tandem dye according to clause 47, wherein R¹is an optionally substituted aryl or heteroaryl linked to anon-conjugated repeat unit of the polymeric backbone.

Clause 49. The polymeric tandem dye according to any one of clauses47-48, wherein Y¹ and Y² each comprise a water solubilizing group (WSG).

Clause 50. The polymeric tandem dye according to any one of clauses47-49, wherein the pendant donor chromophore groups are described by thefollowing structure:

wherein:

* is a point of linkage to a non-conjugated repeat unit of the polymericbackbone;

Y¹ and Y² are each alkynyl substituted with a WSG.

Clause 51. The polymeric tandem dye according to clause 50, wherein thependant donor chromophore groups are described by the followingstructure:

wherein:

R¹¹ is L¹-Z¹ (a linked non-conjugated repeat unit of the polymericbackbone);

each R⁹ is an optional substituent selected from halogen, hydroxyl,cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy,aryl, substituted aryl, heteroaryl and substituted heteroaryl; and t is0-4.

Clause 52. The polymeric tandem dye according to any one of clauses38-51, wherein the pendant donor chromophore groups are substituted withone or more water solubilizing groups (WSGs) independently selected fromthe following formula:

wherein:

T⁵ is an optional linker;

each T⁶ is an linker;

R¹¹ and R are independently H, alkyl or substituted alkyl; and

each s is an integer from 1 to 50.

Clause 53. The polymeric tandem dye according to any one of clauses38-52, comprising a segment of the formula:

wherein:

each M¹ and M² is independently an aryl or heteroaryl co-monomer;

each S¹ and S² is independently a non-conjugated spacer units;

each D¹ is independently a pendant donor chromophore linked to M¹;

each A¹ is independently an acceptor fluorophore linked to M²;

x is 75 mol % or more; and

y is 25 mol % or less.

Clause 54. The polymeric tandem dye according to clause 53, wherein theM¹-S¹ and M²-S² repeat units of the polymeric backbone have a randomconfiguration.

Clause 55. The polymeric tandem dye according to any one of clauses53-54, wherein:

each M¹ and M² independently comprises one or more groups selected fromfluorene, carbazole, silole, biphenylene and phenylene; and

each S¹ and S² is independently a saturated spacer unit selected from adivalent polyethylene glycol (PEG) and a divalent modified PEG group.

Clause 56. The polymeric tandem dye according to any one of clauses38-52, comprising a segment of the formula:

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹ andSM² co-monomers that are each independently a non-conjugated co-monomer;

each D¹ is independently a pendant donor chromophore linked to SM¹;

each A¹ is independently an acceptor fluorophore linked to SM²;

x is 75 mol % or more; and

y is 25 mol % or less.

Clause 57. The polymeric tandem dye according to clause 56, wherein therepeat units of the polymeric backbone have a random configuration.

Clause 58. The polymeric tandem dye according to any one of clauses56-57, wherein SM¹ and SM² are co-monomers derived from an acrylate, amethacrylate, an acrylamide, a polystyrene, a ROMP monomer, an ADMETmonomer or a cyclic carbonate.

Clause 59. The polymeric tandem dye according to clause 58, wherein SM¹and SM² are selected from the following formulae:

wherein:

R²¹ is -L¹-D¹ or -L²-Z¹;

D¹ is the pendant donor chromophore linked to M¹;

Z¹ is a chemoselective tag linked to M²;

L¹ and L² are optional linkers;

X is O or NR″;

R′ is H or lower alkyl;

R″ is H or lower alkyl, substituted lower alkyl and WSG; and

* is a connection to the polymeric backbone.

Clause 60. The polymeric tandem dye according to clause 56, wherein themultichromophore is of formula (XXI):

wherein:

the polymeric backbone of non-conjugated repeat units comprises SM¹, SM²and SM³ co-monomers that are each linked via a group T that is theproduct of a click chemistry or chemoselective group conjugationreaction; SM³ optionally comprises a linked WSG;

each D¹ is independently a pendant light absorbing chromophore linked toSM¹;

each A¹ is independently an acceptor fluorophore linked to SM²;

x is 50 mol % or more; and

y+z is 50 mol % or less, where * is a connection to the polymericbackbone of the multichromophore or a terminal group.

Clause 61. The polymeric tandem dye according to clause 60, wherein SM¹,SM² and SM³ comprise the following structures:

wherein:

each X is independently O or NR³¹ wherein R³¹ is H, alkyl, substitutedalkyl, alkanoyl or substituted alkanoyl;

each r and s is independently 1-6 (e.g., 1, 2 or 3);

each d and e is independently 1-12 (e.g., 1-6, such as 1, 2, 3, 4, 5 or6);

t is 0 or 1;

each L¹, L² and L³ is independently a linker; and

* is a connection to a 1,4-substituted 1,2,3-triazole (T) having one ofthe following structures:

Clause 62. The polymeric tandem dye according to clause 56, wherein therepeat units of the polymeric backbone have a defined linear sequence.

Clause 63. The polymeric tandem dye according to clause 62, wherein SM¹and SM² are co-monomers derived from amino acids, peptoid monomers, aprotected carbonate monomer or a cyclic carbonate monomer.

Clause 64. The polymeric tandem dye according to any one of clauses 56,62 and 63, wherein the polymeric backbone is a polypeptide having adefined sequence of α-amino acid residues and/or β-amino acid residues.

Clause 65. The polymeric tandem dye according to any one of clauses 56and 62-64, wherein the multichromophore is of the formula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor fluorophore;

each L¹ and L² is independently a linker;

p₁ and q₁ are independently 0 or 1 wherein p₁+q₁≤1;

p₂ and q₂ are independently 0 or 1 wherein p₁+q₁≤1;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a donor chromophore group, an acceptor fluorophore, alinker and a linked specific binding member.

Clause 66. The polymeric tandem dye of clause 65, wherein p₁ and p₂ areeach 0 and q₁ and q₂ are each 1.

Clause 67. The polymeric tandem dye of clause 65, wherein p₁ and p₂ areeach 1 and q₁ and q₂ are each 0.

Clause 68. The polymeric tandem dye of clause 65, wherein p₁, p₂, q₁ andq₂ are each 0 and the multichromophore is of the formula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor fluorophore;

L¹ and L² are each independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a donor chromophore group, an acceptor fluorophore, alinker and a linked specific binding member.

Clause 69. The polymeric tandem dye according to any one of clauses 64,65 and 66, comprising a segment of the formula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor fluorophore;

each L¹ and L² are independently a linker;

n and p are each independently an integer from 1 to 20 wherein n+p 2;and

m is 1 or 2.

Clause 70. The polymeric tandem dye according to clause 69, wherein themultichromophore comprises q segments of copolymer and is of theformula:

wherein:

each (n)_(q) and each (p)_(q) is independently an integer from 1 to 20,wherein for each of the q segments (n)_(q)+(p)_(q)≥3; and

q is an integer from 1 to 100.

Clause 71. The polymeric tandem dye according to any one of clauses 56,62-65 and 68-70, wherein the polymeric backbone comprises an amino acidsequence selected from:

XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXXXXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X)_(n)YXY(X)_(n)YX XXY(X)_(n)YXX XXXY(X)_(n)YXXX XXXXY(X)_(n)YXXXXXXXXXY(X)_(n)YXXXXXwherein:

each X is a lysine or ornithine residue covalently N-linked to a pendantdonor chromophore group; and

each Y is a cysteine residue covalently linked to a pendant acceptorfluorophore group.

Clause 72. The polymeric tandem dye according to any one of clauses 56,62 and 63, wherein the multichromophore has the formula:

wherein

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor chromophore;

each L¹ and L² is independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a donor chromophore group, an acceptor fluorophore, alinker and a linked specific binding member.

Clause 73. The polymeric tandem dye according to any one of clauses56-58 and 63, wherein the multichromophore has the formula:

wherein:

each D¹ is independently a pendant donor chromophore;

each A¹ is independently an acceptor fluorophore;

each L¹ and L² is independently a linker;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from the group consisting of aterminal group, a polymer segment, a donor chromophore group, anacceptor fluorophore, a linker and a linked specific binding member.

Clause 74. The polymeric tandem dye according to any one of clauses53-73, wherein the pendant donor chromophore groups are BODIPY groups.

Clause 75. The polymeric tandem dye according to any one of clauses38-74, wherein the acceptor fluorophore (e.g., each A¹) is a smallmolecule fluorophore.

Clause 76. The polymeric tandem dye according to any one of clauses38-75, wherein the acceptor fluorophore (e.g., each A¹) is selected froma cyanine dye, a rhodamine dye, a xanthene dye, a coumarin dye, apolymethine, a pyrene, a dipyrromethene borondifluoride, a napthalimide,a thiazine dye and an acridine dye.Clause 77. A labelled specific binding member, comprising:

a polymeric tandem dye comprising:

-   -   a light harvesting multichromophore comprising:        -   a polymeric backbone comprising non-conjugated repeat units;            and        -   a plurality of pendant donor chromophore groups each            independently linked to a non-conjugated repeat unit of the            polymeric backbone; and    -   an acceptor fluorophore linked to a non-conjugated repeat unit        of the polymeric backbone and configured in energy-receiving        proximity to at least one pendant donor chromophore group of the        light harvesting multichromophore; and

a specific binding member linked to the polymeric tandem dye.

Clause 78. The labelled specific binding member according to clause 77,wherein the specific binding member is an antibody.

Clause 79. The labelled specific binding member according to clause 77,wherein the specific binding member is an antibody fragment or bindingderivative thereof.

Clause 80. The labelled specific binding member according to clause 79,wherein the antibody fragment or binding derivative thereof is selectedfrom the group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv,a diabody and a triabody.

Clause 81. The labelled specific binding member according to any one ofclauses 77-80, wherein the acceptor fluorophore is selected from acyanine dye, a rhodamine dye, a xanthene dye, a coumarin dye, apolymethine, a pyrene, a dipyrromethene borondifluoride, a napthalimide,a thiazine dye and an acridine dye.Clause 82. The labelled specific binding member according to any one ofclauses 77-81, wherein the polymeric tandem dye is a dye according toany one of clauses 39-76.Clause 83. The labelled specific binding member according to any one ofclauses 77-81, having the formula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor fluorophore;

each L¹ and L² are independently a linker;

p₁ and q₁ are independently 0 or 1 wherein p₁+q₁≤1;

p₂ and q₂ are independently 0 or 1 wherein p₁+q₁≤1;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ is a terminal group, a polymer segment, a donor chromophore group, anacceptor fluorophore, or a linker; and

G² is a linked specific binding member.

Clause 84. The labelled specific binding member according to clause 83,wherein p₁, p₂, q₁ and q₂ are each 0 and the multichromophore is of theformula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor fluorophore;

L¹ and L² are each independently a linker;

x is 75 mol % or more; y is 25 mol % or less;

G¹ is a terminal group, a polymer segment, a donor chromophore group, anacceptor fluorophore or a linker; and

G² is a linked specific binding member.

Clause 85. A method of evaluating a sample for the presence of a targetanalyte, the method comprising:

(a) contacting the sample with a labelled specific binding member thatspecifically binds the target analyte to produce a labelling compositioncontacted sample, wherein the labelled specific binding membercomprises:

-   -   (i) a polymeric tandem dye according to any one of clauses        38-76; and    -   (ii) a specific binding member linked to the polymeric tandem        dye; and

(b) assaying the labelling composition contacted sample for the presenceof a labelled specific binding member-target analyte binding complex toevaluate whether the target analyte is present in the sample.

Clause 86. The method according to clause 85, wherein the acceptorfluorophore of the polymeric tandem dye is selected from a cyanine dye,a rhodamine dye, a xanthene dye, a coumarin dye, a polymethine, apyrene, a dipyrromethene borondifluoride, a napthalimide, a thiazine dyeand an acridine dye.Clause 87. The method according to any one of clauses 85-86, furthercomprising contacting the sample with a second specific binding memberthat is support bound and specifically binds the target analyte.Clause 88. The method according to clause 87, wherein the supportcomprises a magnetic particle.Clause 89. The method according to any one of clauses 85-88, wherein thetarget analyte is associated with a cell.Clause 90. The method according to clause 89, wherein the target analyteis a cell surface marker of the cell.Clause 91. The method according to clause 90, wherein the cell surfacemarker is selected from the group consisting of a cell receptor and acell surface antigen.Clause 92. The method according to clause 89, wherein the target analyteis an intracellular target, and the method further comprises lysing thecell.Clause 93. The method according to any one of clauses 85-92, wherein themethod further comprises flow cytometrically analyzing the fluorescentlylabelled target analyte.Clause 94. A method of labelling a target molecule, the methodcomprising:

contacting the target molecule with a polymeric tandem dye to produce alabelled target molecule, wherein:

the polymeric tandem dye is a dye according to any one of clauses 38-76and comprises a conjugation tag that covalently links to the targetmolecule.

Clause 95. The method according to clause 94, wherein the polymerictandem dye has the formula:

wherein:

each D¹ is independently a pendant donor chromophore group;

each A¹ is independently an acceptor fluorophore;

each L¹ and L² are independently a linker;

p₁ and q₁ are independently 0 or 1 wherein p₁+q₁≤1;

p₂ and q₂ are independently 0 or 1 wherein p₁+q₁≤1;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ is a terminal group, a polymer segment, a donor chromophore group, anacceptor fluorophore or a linker; and

G² is a linker comprising the conjugation tag.

Clause 96. A method of preparing a light harvesting multichromophore,the method comprising:

a) synthesizing a protected polypeptide having a defined amino acidsequence consisting of blocks of first amino acid residues separated bysingle occurrences of second amino acid residues, wherein:

each block of first amino acid residues comprises at least two residues;

the first amino acid residues each comprise a protected firstchemoselective sidechain group; and

the second amino acid residues each comprise a protected secondchemoselective sidechain group;

b) deprotecting the protected polypeptide to produce a deprotectedpolypeptide wherein the first and second chemoselective sidechain groupsare deprotected;

c) coupling reactive donor chromophore moieties to deprotected firstchemoselective sidechain groups of the first amino acid residues toproduce pendant donor chromophore groups.

Clause 97. The method according to clause 96, further comprising,sequentially with step c), coupling reactive acceptor fluorophoremoieties to deprotected second chemoselective sidechain groups of thesecond amino acid residues to produce pendant acceptor fluorophores.Clause 98. The method according to clause 96, further comprising, afterstep a), deprotecting the N-terminal of the protected polypeptide andcoupling a G1 group to the N-terminal of the N-terminal deprotectedpolypeptide, wherein G1 is a terminal group (e.g., capping group), adonor chromophore group or a linker.Clause 99. The method according to any one of clauses 96-98, furthercomprising, after step c), coupling a specific binding member to theC-terminal of the polypeptide.Clause 100. The method according to any one of clauses 96-99, whereinthe light harvesting multichromophore is of the formula:

wherein:

D¹ is the pendant donor chromophore group;

Z¹ is the second chemoselective sidechain group;

each L¹ and L² is independently a linker;

p₁ and q₁ are independently 0 or 1 wherein p₁+q₁≤1;

p₂ and q₂ are independently 0 or 1 wherein p₁+q₁≤1;

x is 75 mol % or more;

y is 25 mol % or less; and

G¹ and G² are each independently selected from a terminal group, apolymer segment, a donor chromophore group, an acceptor fluorophore, alinker and a linked specific binding member.

Clause 101. The method according to any one of clauses 96-100, whereinthe light harvesting multichromophore is of the formula:

wherein:

each (n)_(q) and each (p)_(q) is independently an integer from 1 to 20,wherein for each of the q segments (n)_(q)+(p)_(q)≥3; and

q is an integer from 1 to 100.

Clause 102. The method according to any one of clauses 96-101, wherein:

the first amino acid residues are independently selected from lysine andornithine; and

the second amino acid residues are each cysteine.

Clause 103. The method according to any one of clauses 96-102, whereinthe defined amino acid sequence comprises an amino acid sequenceselected from:

XYXX XXYXX XXXYXXX XXXYXXXX XXXXYXXX XXXXYXXXX XXXXXYXXXXX XXXXXXYXXXXXXXXXXXXXYXXXXXXX XXXXXXXXYXXXXXXXX XXXXXXXXXYXXXXXXXXX Y(X)_(n)YXY(X)_(n)YX XXY(X)_(n)YXX XXXY(X)_(n)YXXX XXXXY(X)_(n)YXXXXXXXXXY(X)_(n)YXXXXXwherein:

each X is a lysine or ornithine residue covalently N-linked to a pendantdonor chromophore group; and

each Y is a cysteine residue or a protected cysteine residue.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. Moreover, nothing disclosedherein is intended to be dedicated to the public regardless of whethersuch disclosure is explicitly recited in the claims.

The scope of the present invention, therefore, is not intended to belimited to the exemplary embodiments shown and described herein. Rather,the scope and spirit of present invention is embodied by the appendedclaims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) isexpressly defined as being invoked for a limitation in the claim onlywhen the exact phrase “means for” or the exact phrase “step for” isrecited at the beginning of such limitation in the claim; if such exactphrase is not used in a limitation in the claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

What is claimed is:
 1. A multi-chromophore comprising: a polypeptidebackbone; and a plurality of pendant donor chromophores eachindependently linked to an amino acid residue of the polypeptidebackbone.
 2. The multi-chromophore according to claim 1, wherein thepolypeptide backbone comprises from 2 to 200 amino acid residues.
 3. Themulti-chromophore according to claim 2, wherein the polypeptide backbonecomprises from 4 to 100 amino acid residues.
 4. The multi-chromophoreaccording to claim 1, wherein the polypeptide backbone comprises 50 mol% or more amino acid residues that are independently linked to a pendantdonor chromophore.
 5. The multi-chromophore according to claim 1,wherein the amino acid residues are selected from the group consistingof lysine, glycine, cysteine, aspartate, glutamate and combinationsthereof.
 6. The multi-chromophore according to claim 1, wherein thepolypeptide comprises first, second and third amino acid residues. 7.The multi-chromophore according to claim 6, wherein first amino acidresidue is linked to a donor chromophore, the second amino acid residuecomprises a sidechain-linked second chemo-selective tag and the thirdamino acid residue comprises a spacer residue.
 8. The multi-chromophoreaccording to claim 1, wherein the pendant donor chromophores areselected from a fused tricyclic aryl or heteroaryl group and a BODIPYgroup.
 9. A polymeric tandem dye comprising: a polypeptide backbone; aplurality of pendant donor chromophores each independently linked to anamino acid residue of the polypeptide backbone; an acceptor fluorophorelinked to an amino acid residue of the polypeptide backbone andconfigured in energy-receiving proximity to at least one pendant donorchromophore of the plurality of pendant donor chromophores.
 10. Thepolymeric tandem dye according to claim 9, wherein the polypeptidebackbone comprises from 4 to 100 amino acid residues.
 11. The polymerictandem dye according to claim 9, wherein the amino acid residues areselected from the group consisting of lysine, glycine, cysteine,aspartate, glutamate and combinations thereof.
 12. The polymeric tandemdye according to claim 9, wherein the pendant donor chromophores areselected from a fused tricyclic aryl or heteroaryl group and a BODIPYgroup.
 13. The polymeric tandem dye according to claim 9, wherein theacceptor fluorphore is a dye molecule selected from a rhodamine, acoumarin, a xanthene, a cyanine, a polymethine, a pyrene, a thiazine, anacridine, a dipyrromethene borondifluoride, a napthalimide, aphycobiliprotein, a peridinum chlorophyll protein, conjugates thereof,and combinations thereof.
 14. The polymeric tandem dye according toclaim 9, wherein the number of pendant donor chromophores is greaterthan the number of acceptor fluorophores.
 15. The polymeric tandem dyeaccording to claim 14, wherein the ratio of pendant donor chromophoresto acceptor fluorophores ranges from 5:1 to 10:1.
 16. The polymerictandem dye according to claim 14, wherein the polypeptide backbonecomprises 50 mol % or more amino acid residues that are independentlylinked to a pendant donor chromophore.
 17. The polymeric tandem dyeaccording to claim 15, wherein 1 mol % to 25 mol % of the amino acidresidues are linked to an acceptor fluorophore.
 18. A labelled specificbinding member, comprising: a polymeric tandem dye comprising: apolypeptide backbone; a plurality of pendant donor chromophores eachindependently linked to an amino acid residue of the polypeptidebackbone; an acceptor fluorophore linked to an amino acid residue of thepolypeptide backbone and configured in energy-receiving proximity to atleast one pendant donor chromophore of the plurality of pendant donorchromophores; and a specific binding member linked to the polymerictandem dye.
 19. The labelled specific binding member according to claim18, wherein the specific binding member is an antibody, an antibodyfragment or binding derivative thereof.
 20. The labelled specificbinding member according to claim 18, wherein the polypeptide backbonecomprises from 4 to 100 amino acid residues.
 21. The labelled specificbinding member according to claim 18, wherein the amino acid residuesare selected from the group consisting of lysine, glycine, cysteine,aspartate, glutamate and combinations thereof.
 22. The labelled specificbinding member according to claim 18, wherein the pendant donorchromophores are selected from a fused tricyclic aryl or heteroarylgroup and a BODIPY group.
 23. The labelled specific binding memberaccording to claim 18, wherein the acceptor fluorophore is a dyemolecule selected from a rhodamine, a coumarin, a xanthene, a cyanine, apolymethine, a pyrene, a thiazine, an acridine, a dipyrrometheneborondifluoride, a napthalimide, a phycobiliprotein, a peridinumchlorophyll protein, conjugates thereof, and combinations thereof. 24.The labelled specific binding member according to claim 18, wherein thenumber of pendant donor chromophores is greater than the number ofacceptor fluorophores.
 25. The labelled specific binding memberaccording to claim 24, wherein the ratio of pendant donor chromophoresto acceptor fluorophores ranges from 5:1 to 10:1.
 26. The labelledspecific binding member according to claim 24, wherein the polypeptidebackbone comprises 50 mol % or more amino acid residues that areindependently linked to a pendant donor chromophore.
 27. The labelledspecific binding member according to claim 25, wherein 1 mol % to 25 mol% of the amino acid residues are linked to an acceptor fluorophore.